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TGGE System

1. The calculation of the corresponding temperatures is simple since there is a linear temperature gradient between L1 and L6 i e the temperature increment from one to the next line is always the same Calculation Divide range of gradient by five L6 L1 this is the temperature increment from one line to the adjacent lane Example calculation of temperature at line 2 5 in a temperature gradient from 40 C L1 to 60 C L6 e subtract temperature at L1 from temperature L6 range of gradient 60 40 C 20 C e divide temperature by 5 increment per lane 20 C 5 4 C e multiply increment by 1 5 1 5 increments from L1 to L2 5 6 C e add this value to the temperature at L1 40 C 6 C Result temperature at L 2 5 is 46 C TGGE Manual October 2009 36 Biometra An Analytik Jena Company 6 4 Programming the TGGE Controller All parameters of the run are controlled by the TGGE system controller This includes electrophoretic parameters voltage amperage time as well as control of the temperature gradient After switching on the controller the display shows the instruments name and software version Then the main menu appears Note Each program can consist of several steps Thus you can program pre run equilibration and main run in the same program 6 4 1 Software main menu T1 22 0 C T2 22 0 C At the bottom line the display shows 4 options which can be activated by the 4 functions keys A
2. Figure 9 Components of the TGGE cuvette for casting gels Note The gel is poured on Polybond film to optimize temperature transition between block and gel The gel is bound covalently to this hydrophilic Polybond film 024 030 Note The gel sticks to the Polybond film throughout the whole procedure including staining 1 Glass plates must be dry and free of any dirt or dust Please wear powder free gloves during cleaning and assembly of gel sandwich to prevent contamination which will interfere with silver staining Clean both glass plates with 70 ethanol and a soft tissue TGGE Manual October 2009 25 3 4 Biometra An Analytik Jena Company Do not use strong acidic or basic solutions or organic solvents for cleaning the glass plates Do not apply mechanical stress on slot formers i e brushing or rubbing Do not incubate glass plates over night in cleaning solutions The glass plate with spacer and slot former must be carefully treated with a repelling agent This is essential for easy removal of the polymerized gel from the glass plate with slot formers Apply approx 0 5 ml of gel repellent solution Acryl Glide Amresco or Repel silane Sigma on the glass plate WITH SPACERS and spread it carefully especially between the slot former Wait 5 min than polish the plate with soft tissue to remove residual solution A A Clean spacers with ethanol before assembling the glass plate sandwich Do
3. la disposition des utilisateurs finaux en cons quence des r gulations par la loi de votre pays Pour des information additionel nous Vous demandons de contacter votre distributeur Cet symbole s applique uniquement aux pays de EEE EEE Espace conomique europ en qui regroupe les Etats membres de TUE et la Norv ge Islande et le Liechtenstein TGGE Manual October 2009 56 Biometra An Analytik Jena Company 14 EU Konformitatserklarung EU Declaration of Conformity Gottingen den 01 10 1997 im Sinne der EG Richtlinie ber elektrische Betriebsmittel zur Verwendung innerhalb bestimmter Spannungsgrenzen 73 23 EWG Anhang III following the EC directive about electrical equipment for use within certain limits of voltage 3 23 EWG appendix 3 und and im Sinne der EG Richtlinie fur die elektromagnetische Vertr glichkeit 89 336 EWG Anhang I following the EC directive about theelectromagnetic compability 89 336 EWG appendix 1 Hiermit erkl ren wir da folgende Elektrophoresegerate Herewith we declare that the following gel electrophoresis systems Typen TGGE System TGGE Stromversorgungsger t mit integriertem Controller TGGE Elektrophoreseeinheit mit Peltier Element TGGE Pufferkammer types TGGE system TGGE power supply with integrated controller TGGE electrophoresis unit with Peltier element powered gradient block TGGE electrophoresis chamber Best Nr Order No 024 000 024 001 024 002
4. 19 2993 3000 Po Tien Steger G Rosenbaum V Kaper J and Riesner D 1987 Double stranded cucumovirus associated RNAS experimental analysis of nec rogenic and non necrogenic variants by temperature gradient gel electrophoresis Nucleic Acids Res 15 5069 5083 Zimmat R Gruner R Hecker R Steger G and Riesner D 1991 Analysis of mutations in viroid RNA by non denaturing and temperature gradient gel electrophoresis In R H Sarma and M H Sarma eds Structure amp Methods Vol 3 DNA amp RNA Adenine Press 339 357 Rosenbaum V Klahn T Lundberg Holmgren E von Gabain A and Riesner D 1992 Co existing structures of an MRNA stability determinat The 5 region of the Escherichia coli and Serratia marcescens ompA mRNA J Mol Biol in press Birmes A Sattler A Maurer S O and Riesner D 1990 Analysis of the conformational transitions of proteins by temperature gradient gel electrophoresis Electrophoresis 11 795 801 Sattler A Kanka S Schr rs W and Riesner D 1992 Random mutagenesis of the weak calcium binding side in SubtilisinCarlsberg and screening for thermal stability by temperature gradient gel electrophoresis Accepted for 17 International Symposium of Subtilisin Enzymes EMBL Hamburg Thatcher D and Hodson B 1981 Denaturation of proteins and nucleic acids by thermal gradient electrophoresis Biochem J 197 105 109 Wagenhofer M Hansen D and Hillen W 1988
5. C quit D enter lf the program name is complete confirm name with D enter In the following screen you can set the temperatures for the gradient block 6 4 5 Enter temperatures for the gradient block 1 L0 L6 A B CquitD Enter temperature for LO and accept with D enter 1 L0 30 0 L A Bdelete Cquit Denter Enter temperature for L6 and accept with D enter 1 L0 30 0 L6 70 0 ok A Bno Cauit Dyes Confirm settings with D yes If settings are not correct press B no and repeat entry of temperature settings After you have confirmed the temperature settings the following screen is displayed Here you can enter all parameters for electrophoresis 6 4 6 Enter electrophoresis parameters 1 L0 25 0 L6 60 0 time El OV 500mA 30W A BV h C quit D gt Enter time for electrophoresis and accept with D enter TGGE Manual October 2009 38 Biometra An Analytik Jena Company Note There is a convention on how time settings are entered in all BIOMETRA instruments hours e minutes e seconds lf you enter a number without dot this value will be interpreted as seconds 300 gt 5 minutes To program minutes enter a e after the number of minutes To enter hours enter e e after the number You can also enter any combination of hours minutes and seconds Example for 1 hour 30 minutes 20 seconds enter 1 30 e 20 The time values will be displayed in the following format 00 m 00s Accept time
6. Kuhn J E et al 1995 Quantitation of human cytomegalovirus genomes in the brain of AIDS patients Journal of Medical Virology 47 70 82 7 Linke B et al 1995 Identification and structural analysis of rearranged immunoglobulin heavy chain genes in lymphomas and leukemia Leukemia 9 840 847 8 Menke M A et al 1995 Temperature gradient gel electrophoresis for analysis of a polymerase chain reaction based diagnostic clonality assay in the early stages of cutaneous T cell lymphomas 9 Hecker R et al 1988 Analysis of RNA structure by temperature gradient gel electrophoresis viroid replication and processing Gene 72 59 74 10 Baumstark T and Riesner D 1995 Only one of four possible secondary structures of the central conserved region of potato spindle tuber viroid is a substrate for processing in a potato nuclear extract Nucleid Acids Research 23 4246 4254 11 Loss P Schmitz M Steger G and Riesner D 1991 Formation of a thermodynamically metastable structure containing hairpin II is critical for the potato spindle tuber viroid EMBO Journal 10 719 728 12 Riesner D 1998 Nucleic acid structures In Antisense Technology Practical Approach Series Oxford University Press p1 24 in press 13 Wiese U et al 1995 Scanning for mutations in the human prion protein open reading frame by temporal temperature gradient gel electrophoresis Electrophoresis 16 1851 1860 14 Nubel U et al 199
7. Therefore extended migration times and higher voltage will rather lead to fuzzy bands than to a better resolution To improve resolution i e distance between two bands in one lane the temperature gradient has to bee adjusted see section 8 1 In general migration of samples in TGGE depends on e Temperature gradient e Buffer system Gel composition e Voltage e The kind of sample e g protein nucleic acid fragment size e The kind of application e g parallel or perpendicular TGGE e Sample preparation e g high salt or low salt preparation Any recommendations should be regarded as guidelines to start with Further improvement of the analysis should be done by adjusting the run conditions to individual needs Important The TGGE controller is designed to control voltage NOT amperage mA and Vh should be set to maximum 6 3 2 Conditions for mutation analysis Since each DNA fragment has its own melting characteristic there can be no general valid parameters for mutation analysis The following parameters have been optimized for separating the Biometra TGGE test kit DNA 024 050 If other fragments are to be analyzed please optimize parameters as described in chapter 4 and 8 Gel composition 1 x TAE buffer 8 Polyacrylamide 37 5 1 8M Urea 2 Glycerol Temperature gradient L1 39 C L6 54 C Electrophoresis 250V 50 min TGGE Manual October 2009 30 Biometra An Analytik Jena Company 6 3 3 Conditions for d
8. perpendicular TGGE D l l Optimize primer design No separation of hetero duplex Inappropriate fragment Perform Poland analysis ACE Wrong temperature gradient Perform perpendicular TGGE Acrylamide of poor quality Use only high quality chemicals p a Irreproducible gels Erratic temperature Use only minimum volume of thermal distribution over and under coupling solution under the gel the gel Do not overlay gel with buffer Silver staining Bad silver stain Chemicals of poor quality Use only high quality chemicals Stale water Use only freshly prepared aqua bidest Too much silver nitrate Refer to the staining protocol TGGE Manual October 2009 44 Biometra An Analytik Jena Company Strong background Insufficient washing after Extend wash step change water incubation in staining frequently solution Weak staining of bands Excessive washing after Reduce wash step after staining binding of staining solution 8 1 Error messages TGGE connector cable is not connected to gradient block and or TGGE System check connection System Controller Check connections to thermoblock Warning Invalid gradient temperatures maximum gradient 45 C Gradient too large max grad T1 gt T2 45 C A Bno gt L1 C quit Denter Warning Invalid gradient temperatures maximum gradient 45 C Gradient too large max grad T1 gt T2 45 C A Bno gt T1 C quit Denter It is possible to review a program during
9. 020 010 024 090 024 091 024 092 den grundlegenden Anforderungen der corresponds to the basic requirements of EG Niederspannungsrichtlinie 73 23 EWG i d F 93 68 EWG und der EC low voltage directive 73 23 EWG in version 93 68 EWG and the EG Richtlinie ber die elektromagnetische Vertr glichkeit 89 336 EWG i d F 93 68 EWG entsprechen EC directive about the electromagnetic compatibility 89 336 EWG in version 93 68 EWG Folgende harmonisierte Normen wurden angewandt The following harmonized standards have been used EN 50081 1 EN 50082 1 EN 60555 2 EN 60555 3 EN 61010 1 EN 61010 2 Quality Manager Dr J rgen Otte TGGE Manual October 2009 57 Biometra An Analytik Jena Company 15 Warranty This Biometra instrument has been carefully build inspected and quality controlled before dispatch Hereby Biometra warrants that this instrument conforms to the specifications given in this manual This warranty covers defects in materials or workmanship as described under the following conditions This warranty is valid for 24 month from date of shipment to the customer from Biometra This warranty will not be extended to a third party without a written agreement of Biometra This warranty covers only the instrument and all original accessories delivered with the instrument This warranty is valid only if the instrument is operated as described in the manual Biometra will repair or replace each part which is returned and found to
10. For example in parallel TGGE nonspecific bands with a higher molecular weight than the specific PCR product may be misinterpreted as heteroduplices or analogs with lower thermal stabilities Therefore prior to TGGE check the PCR product in a conventional agarose gel If necessary purify your specific PCR product e g by agarose gel electrophoresis and subsequent gel extraction 5 2 Sample preparation for direct DNA analysis 1 volume of DNA RNA samples is mixed with 1 volume of TBE or Na TAE loading buffer or with 0 1 volume of the total loading volume ME loading buffer see Appendix The resulting mixture is loaded directly on to the polyacrylamid gels Be sure that the slots are filled up to maximum if necessary add 1x loading buffer to fill up the slots to maximum In case of low concentration samples we recommend to prepare 5x conc loading buffer 0 2 volume of this concentrated loading buffer is mixed with 0 8 volumes of the sample and loaded onto the gel 5 3 Denaturation Renaturation for heteroduplex analysis of DNA Mix sample with equal amount of standard DNA and heat to 95 C for 5 minutes denaturation Then let slowly cool down to 50 C renaturation This can be done by programming a thermocycler to 94 C for 5 minutes and then 50 C for 15 minutes with a ramping rate of 0 1 C second The sample is then loaded directly to the gel In order to achieve the recommended loading volumes for diagonal or perpendicular TGG
11. Remove gel from 10 The gel will dry completely to the gel support storage glycerol and let it dry film and can then be stored indefinitely 7 2 Ethidium bromide staining Incubate the gel in staining solution 0 5 ug ml ethidium bromide in 1 X TAE for 30 45 min Analyze under UV radiation 27 Note The TGGE gel must be positioned face down on the UV table Otherwise the support film will shield bands from excitation by UV light 7 3 Autoradiography TGGE gels can also be directly exposed to x ray films if radiolabeled samples are analyzed Direct exposure Incubate the TGGE gel for 15 min in Fixation solution see 6 5 Silver staining Optional Silver stain the gel Remove residual buffer from the gel Expose to an x ray film at room temperature Exposure of dried TGGE gels Incubate the TGGE gel for 15 min in Fixation solution see 6 5 Silver staining Optional Silver stain the gel Incubate the gel in 2 5 glycerol for 10 minutes to prevent the gel from cracking Incubate an on the gel Air dry at room temperature for one day or use a gel dryer at 50 C for at least 3h Expose to an X ray film 7 4 Elution of DNA from the TGGE gel DNA fragments which have been separated on TGGE for example different alleles of one gene can be eluted from silver stained TGGE gel and re amplified by PCR Using a Pasteur pipette puncture the gel and extract a ul piece containing the particular DNA duplex Incubate in 20 ul TE buf
12. Thermal denaturation of engineered tet repressor proteins and their complexes with tet operator and tetracycline studie by temperature gradient gel electrophoresis Analytical Biochem 175 422 432 Sanguinetti C J Neto E D and Simpson A J G 1994 BioTechniques 17 915 Kappes S Milde Langosch K Kressin P Passlack B Dockhorn Dwornczak B R hlke P and L ning T 1995 p53 Mutations in ovarian tumors detected by temperature 45 gradient gel electrophoresis direct sequencing and immunohistochemistry Int J Cancer 64 52 59 Kluwe L MacCollin M Tatagiba M Thomas S Hazim W Haase W and Mautner V F 1998 Phenotypic variability associated with 14 splice site mutations in the NF2 46 gene American Journal of Medical Genetics 77 228 233 Lerman L S and Beldjord C 1998 Comprehensive mutation detection with denaturing gradient gel electrophoresis In R G H Cotton E Edkins and S Forrest eds Mutation 47 Detection A Practical Approach Oxford University Press 35 62 Gamper H Piette J and Hearst J E 1984 Photochem Photobiol 40 29 ff TGGE Manual October 2009 61 Biometra An Analytik Jena Company 16 2 Buffers 16 2 1 Running buffers TBE Running buffer 0 1 x conc TBE up to 1x conc TBE is possible 10 x TBE stock solution 890 mM Boric Acid 20 mM EDTA 890 mM TRIS Do not titrate to adjust pH TAE Running Buffer 1 x conc TAE p
13. be defective This warranty does not apply to wear from normal use failure to follow operating instructions negligence or to parts altered or abused TGGE Manual October 2009 58 Biometra An Analytik Jena Company 16 Appendix 16 1 References Note To get an updated overview of all publications available on TGGE please go to the website of Medline http www ncbi nlm nih gov PubMed and enter the keyword TGGE 1 Riesner D Henco K and Steger G 1990 Temperature Gradient Gel Electrophoresis A method for the analysis of conformational transitions and mutations in nucleic acids and protein Page 169 250 In Chrambach A Dunn M J Radola B J Advances in Electrophoresis Vol 4 VCH Verlagsgesellschaft Weinheim 2 Kappes S et al 1995 p53 mutations in ovarian tumors detected by temperature gradient gel electrophoresis direct sequencing and immunohistochemistry Int J Cancer 64 52 59 3 Milde Langosch K et al 1995 Presence and persistence of HPV and p53 mutation in cancer of the cervix uteri and the vulva Int J Cancer 63 639 645 4 Horn D et al 1996 Three novel mutations of the NF1 gene detected by temperature gradient gel electrophoresis of exons 5 and 8 Electrophoresis 17 1559 1563 5 Wieland U et al 1996 Quantification of HIV 1 proviral DNA and analysis of genomic diversity b ypolymerase chain reaction and temperature gradient gel electrophoresis J Virology Methods 57 127 139 6
14. buffer wicks binder clips sample Acryl Glide only in Germany 4 Manual Please keep the original packaging material for return shipment in case of servicing TGGE System 230 115 V electrophoresis unit with high precision gradient block 2 buffer chambers for variable positioning controller with integrated power supply manual starter kit 024 003 024 000 TGGE System 230 115 V electrophoresis unit with high precision gradient block 2 buffer chambers for variable positioning controller with integrated power supply manual starter kit 024 093 024 090 3 2 System Overview The TGGE System contains all components necessary to get started All kinds of TGGE applications parallel or perpendicular TGGE Constant Temperature GE Time resolved TGGE can be run with the Biometra TGGE System A broad range of different glass plates are available and can be ordered from Biometra or your local distributor see section 10 TGGE Manual October 2009 13 Biometra An Analytik Jena Company Safety lid Hec trophoresis unit controller cord plug Figure 3 TGGE Electrophoresis unit 3 3 Unpack and Check Unpack and carefully examine the instrument Report any damage to Biometra Do not attempt to operate this device if physical damage is present Please keep the original packing material for return shipment in case of service issues A I Attention A Please fill out and send back the warranty re
15. covered with a gel cover film 024 032 The complete setup consisting of gel with cover film and buffer wicks is covered with the gel cover plate The gel cover plate has two sealings and fits tightly onto the thermoblock It holds the buffer wicks in place and helps to build a humidity chamber around the gel This is important to prevent evaporation during the run Important Never run a gel without gel cover plate This could lead to massive condensation under the safety lid Danger of electric shock Gel cover plate Y support film Sample gel cover film buffer wick buffer wick thermoblock Figure 14 Set up of the gel for electrophoresis TGGE Manual October 2009 31 Biometra An Analytik Jena Company 6 3 5 Prior to assembly of the electrophoresis unit Note Be sure to have everything on hand to avoid extended handling times Do not let the disassembled gel dry while setting up the electrophoresis system Prepare e parallel or perpendicular gel see section 6 3 7 or section 6 3 8 e samples in loading buffer for sample preparation see chapter 6 4 e 500 ml 1 x TAE running buffer chapter 16 2 1 e 2 buffer wicks 024 003 e 1 cover film 024 035 e thermal coupling solution 0 01 Triton e gel cover plate 024 031 6 3 6 Gel setup for electrophoresis 1 Adjust electrophoresis chamber with the 4 levelling feet 2 Fill 250 ml running buffer in each buffer tank chec
16. each sample with an external standard In most cases this standard is a PCR fragment without mutations for example amplified from the wild type After mixing the standard DNA fragment with the PCR fragment from the sample the mixture is heated and subsequently slowly cooled down for protocol see section 5 3 a a A A a a a A A a A A gp denature SS SS SC E _ 717 17 I Z E re anneal mutant wildtype homoduplex heterod oices homoduplex sample standard mutant P wildtype Figure 20 Principle of heteroduplex analysis The re annealing of sample and standard results in 4 different DNA fragments 1 The wildtype homoduplex AA 2 the mutant homoduplex aa 3 and 4 two different heteroduplices Aa and aA These heteroduplices carry at least one mismatch disturbed base pairing and have a significant lower melting temperature than the homoduplices This procedure results in a complete denaturing of both double stranded PCR fragments and a subsequent re annealing If the sample is different from the standard re annealing leads to 4 different double stranded DNA fragments see Figure 20 1 the homoduplex of the standard wildtype AA 2 the homoduplex of the sample mutant aa 3 and 4 two heteroduplices between standard and sample Aa and aA Due to the differences between sample and standard these heteroduplices display mismatches in their base pairing in least one position Such mismatches have a strong impac
17. integrate a GC clamp into a PCR fragment one of the two primers has to be modified The non specific GC sequence is added to the 5 end of the primer Thus the GC sequence is incorporated in the fragment during PCR Prior to order primers check the melting behaviour of the PCR fragment with GC clamp by Poland analysis see chapter 4 2 1 Figure 6 shows a Tm plot for a PCR fragment with GC clamp either at the 5 or 3 end GC clamp at 5 end GC clamp at 3 end fia Gees fia cakes SSC orl onda NEEN Kb Tic 100 100 bubble A so so o l 50 Lad L50 50 LO 150 Sequence f nt Sequence nt Figure 6 Tm Plot of a PCR fragment with GC clamp at the 5 end or at the 3 end Whereas the fragment with 5 GC clamp has domains that melt consecutively and is well suited for TGGE analysis the fragment with 3 GC clamp has a domain in the middle that melts at lower temperature compared to the neighbouring domains and is therefore leading to bubble formation Thus the 3 GC clamp for this fragment will not work for TGGE analysis 4 2 3 Chemical clamp with Psoralen Furo 3 2 g coumarin C HsO In addition to clamping a fragment with an artificial high melting domain it is as well possible to covalently fix the end of a PCR fragment To achieve this one of the primers carries a Psoralen molecule Psoralen is a high reactive group when exposed to UV radiation Thus it is possible to covalently close one end of the PCR fragment The
18. of Is gc_15_25_ 34 389 on September Lode Ji OGO Stack index vs Temp of 50 probability first order second order ag 8 Ei fa CU 6J m CG no RA aed j el 5 a UN e Ta CH ua K N CH 60 Temp o 2 SR BO OU 100 110 120 130 140 Figure 5 Tm plot of a 140bp DNA fragment resulting from Poland analysis The second order curve red color in the original shows two different melting domains lf the fragment consists of a single melting domain only or if you want to scan the entire fragment for mutations add a so called GC clamp to one end of the PCR fragment TGGE Manual October 2009 20 Biometra An Analytik Jena Company 4 2 2 GCclamps A GC clamp is an artificial high melting domain which is attached to one end of the fragment during PCR The name GC clamp implies that this short stretch will hold the DNA fragment together preventing a dissociation into the single strands at higher temperatures The optimum location for the GC clamp at the PCR fragment 5 or 3 can be easily checked with the Poland software Copy paste the GC sequence to either side of your sequence and repeat Poland analysis In the following box you will find different examples for a GC clamp short GC clamp 23 bp cccgc cgcgc cccgc cgccc gcc long GC clamp 40 bp 44 cgccc gecge gcccc gegcc cggcc cgccg ccecce gcccg long GC camp 39 bp 45 ccccg ccecce gccge ccecce ccgcg cccgg cgccc ccge To
19. run After pressing SC Program no TEST Pgm is active Program name and store is displayed A copy B del C quit D display Program no __ This program number has not been programmed Name not programmed AT BY Caquit Denter L6 No temperature or time settings entry required T1 T2 A BL C quit D No temperature or time settings entry required A BT1 Coup D gt TGGE Manual October 2009 45 Biometra An Analytik Jena Company 8 2 Optimization of parallel TGGE To improve separation in parallel TGGE the gradient should start directly at the temperature where the fragments start to melt see perpendicular gel and should be rather flat This means there should be only a moderate temperature increase over the whole gel Different fragments in one sample separate as soon as the first fragment starts to melt At a certain higher temperature the next fragment starts to melt In a moderate gradient the temperature increase per centimeter is smaller than in a steeper gradient This means the distance between two temperatures i e locations in the gel is bigger than in a steeper temperature gradient This results in a wider separation of fragments that melt at different temperatures see Figure 19 A DDDDDDD 30 C dereen 2 D SS 2 S SE Deg I Go 70 C TTT ev B oo0o0r00200 0 30 C Rassen nenn 2 D O u a SE II Lg AO ef y Figure 19 Parallel TGGE using a
20. 2009 22 Biometra An Analytik Jena Company 4 4 Parallel analysis of multiple samples After identification of T1 and T2 in a perpendicular TGGE this temperature gradient is spread over the whole block for parallel analysis electrophoresis T1 T2 cold warm Figure 8 Application of T1 and T2 in a parallel gel Note The DNA fragments are separated by their melting behavior They can be distinguished as soon as the fragments begin to melt i e they form a fork like structure temperature higher than T1 During electrophoresis the fragments should not separate into single strands This is an irreversible transition resulting in diffuse bands Note If there are only small differences in the migration of different samples perform a heteroduplex analysis see chapter 8 4 TGGE Manual October 2009 23 Biometra An Analytik Jena Company 5 Sample preparation 5 1 Purity of samples Due to the high sensitivity of the staining procedure after TGGE it is recommended to use purified DNA RNA or protein samples Any impurities might be misinterpreted after TGGE thereby making the analysis of gels difficult Nevertheless it is possible to use even crude mixtures for TGGE analysis PCR amplified DNA fragments usually can be analyzed without further purification Please note that the presence of high amounts of nonspecific secondary PCR products may result in difficulties with interpretation of band pattern melting profile etc
21. 6 Hecker R Wang Z Steger G and Riesner D 1988 Analysis of RNA structure by temperature gradient gel electrophoresis viroid replication and processing Gene 72 59 74 Jiang L Chen W Tain L P and Liu Y 1991 Temperature gradient gel electrophoresis of apple scar skin viroid Acta Microbiol Sin 30 278 283 Riesner D Hecker R and Steger G 1988 Structure of viroid replication intermediates as studied by thermodynamics and temperature gradient gel electrophoresis In Sarma R H and Sarma M H eds Structure amp Expression Vol I From Proteins to Ribosomes Adenine press 261 285 TGGE Manual October 2009 60 33 34 35 36 37 38 39 AO 41 42 43 44 Biometra An Analytik Jena Company Riesner D Steger G Zimmat R Owens R A Wagenh fer M Hillen W Vollbach S and Henco K 1989 Temperature gradient gel electrophoresis of nuleic acids Analysis of confor mational transitions sequence variations and protein nucleic acid interactions Electrophoresis 10 377 389 Rosenbaum V and Riesner D 1987 Temperature gradient gel electrophoresis thermodynamic analysis of nucleic acids and proteins in purified form and in cellular extract Biohys Chem 26 235 246 Sch nborn J Oberstra J Breyel E Tittgen J Schumacher J Lukacs N 1991 Monoclonall antibodies to double stranded RNA as probes of RNA structure in crude nucleic acid extracts Nucleic Acids Res
22. 6 Sequence heterogenities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis TGGE Manual October 2009 59 15 16 17 18 19 20 21 22 23 24 20 26 21 28 29 30 31 32 Biometra An Analytik Jena Company Lessa E P and Applebaum G 1993 Screening techniques for detecting allelic variation in DNA sequences Molecular Ecology 2 119 129 Richter A Plobner L Schumacher J 1997 Quantitatives PCR Verfahren zur Bestimmung der Plasmidkopienzahl in rekombinanten Expressionssystemen BlOforum 20 545 547 Henco K and Heibey M 1990 Quantitative PCR the determination of template copy numbers by temperature gradient gel electrophoresis Nucleic Acids Research 18 6733 6734 Birmes A et al 1990 Analysis of the conformational transition of proteins by temperature gradient gel electrophoresis Electrophoresis 11 795 801 Arakawa T et al 1993 Analysis of the heat induced denaturation of proteins using temperature gradient gel electrophoresis Analytical Biochemistry 208 255 259 Chen X et al 1995 High resolution SSCP by optimization of the temperature by transverse TGGE Nucleic Acids Research 23 4524 4525 Scholz R B et al 1993 Rapid screening for Tp53 mutations by temperature gradient gel electrophoresis a comparison with SSCP analysis Human Molecular Genetics 2 2155 2158 Elphinstone a
23. B C D Block off These 4 options change during programming relative to the chosen menu A BElpho C programs D A retrieve comments or tips about the current program step B Elpho commands to open and start a program C programs edit existing or create new programs D further options like printing of programs or protocols choice of language selecting signal 6 4 2 Create edit program Press C programs to enter the programming mode Program no Alist Bdel Cquit Denter 6 4 3 Select program Enter a program directly by number or press A list to view a list of the existing programs 0 test 1 1 parallel 2 empty AA BY C quit D enter Scroll through the list with AM BY and accept highlighted program with D enter 6 4 4 Name program Each program is specified by name and a program number To facilitate retrieval of a program you can enter a name for each program existing of letters numbers and symbols name gt lt TGGE Manual October 2009 37 Biometra An Analytik Jena Company ABCDEFGHIJKLMNOPQRST UVWXYZ ad amp A gt BABC C quit D enter Press B ABC to enter the mode for the selection of letters name gt lt ABCDEFGHIJKLMNOPQRST UVWXYZ ad amp A B gt C quit D enter Move to the desired letter with A and B gt Accept highlighted letter with D enter name gt test lt ABCDEFGHIJKLMNOPQRST UVWXYZ a O amp A B gt
24. Biometra An Analytik Jena Company TGGE System Instruction Manual Ver 09 10 Model Order No TGGE System 024 000 TGGE System international 024 090 Please read these instructions carefully before using this apparatus Biometra GmbH Rudolf Wissell Str 30 Service Department D 37079 Gottingen Rudolf Wissell Strasse 14 16 Tel 49 0 551 50 68 6 0 D 37079 G ttingen Fax 49 0 551 50 68 6 66 Tel 49 0 551 50 68 6 10 or 12 email Info biometra com Fax 49 0 551 50 68 6 11 Internet http www biometra com email Service biometra com Biometra An Analytik Jena Company This document describes the state at the time of publishing It needs not necessarily agree with future versions Subject to change TGGE Manual October 2009 2 Biometra An Analytik Jena Company Contents Me Ta te e TL 1 PRRERRERELPBENEERERSEINEEEERDNESERNPEHNEACBEEEEEEFEEEUUEEEENEEEEPEDEEGERSBER BESCEUB EEESEPEENEPEFUREREPESRERAENE 6 1 1 Principle Ol the IN GINO 6 1 2 PEGO el CONS EE 6 1 3 SS Cl SU OS ee ee er es en 7 1 3 1 Peltier powered linear temperature gradient nenn nenn nennen 7 1 3 2 SET IY RAN ccrs A E E 8 1 3 3 Paen d ne leie y aaa eee Sn ee ee 8 1 4 Reuler Eeer er le E 9 1 5 Bebe 10 1 5 1 ele TNA E 10 1 5 2 Bei EE 10 1 5 3 Meaning of the Instruchons neuen nnnnnnnnnn nenn ennnne nennen 10 2 Safety and Warning Notices euuuss an000nnnnnnunnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnunnnnnnnnnnnnnnnnnnn 11 2 1 DEINII
25. E the sample volume should be adjusted with running buffer Important Do not denature renature DNA for community profiling see chapter 6 3 3 TGGE Manual October 2009 24 Biometra An Analytik Jena Company 6 Operating 6 1 Casting of gels 6 1 1 Assembly of the gel cuvette The TGGE system 024 000 contains all components needed for casting custom gels glass plates clamps etc There are two different types of gels for TGGE For perpendicular gels one long slot is used to separate one sample over a broad temperature range use glass plate with slot former 024 023 For parallel gels samples are applied to single slots use glass plate with slot formers 024 022 or 024 025 The gel cuvette consists of one glass plate with spacers and slot formers 024 022 or 024 025 and a glass plate without spacers 024 021 The sandwich is fixed with 3 plastic clamps The thinness of the polyacrylamide gel makes it necessary to cast the gel on a gel support film Polybond film order number 024 030 The gel will stay on this film throughout the whole procedure including silver stain A Cover glass plate without spacers bonding plate B Polybond film C Glass plate with slot formers and spacers different types of slots available Bonding plate 024 021 Glass plate with slot fomer and spacers 4 02 Poybond support film 024 030
26. H 8 0 50 x TAE stock solution 242g Tris base 2M 10 x conc pH 8 0 57 1 ml glacial acid 100ml 0 5M EDTA pH 8 0 MOPS Running Buffer 1 x conc MOPS 50x MOPS stock solution 1M MOPS MOPS EDTA ME 50 x conc 50 mM EDTA pH 8 0 16 2 2 Loading buffers Loading buffer TBE TBE running buffer 0 1 Triton X 100 0 01 Bromophenol Blue dye 0 01 Xylene Cyanol dye Loading buffer TAE TAE running buffer 0 1 Triton X 100 0 01 Bromophenol Blue dye 0 01 Xylene Cyanol dye 2mM EDTA TGGE Manual October 2009 62 Loading buffer MOPS 16 3 Other buffers TE buffer TEMED APS Glycerol 40 Glycerol 50 TGGE Manual October 2009 Biometra An Analytik Jena Company MOPS running buffer 1mM EDTA 0 05 Bromophenol Blue dye 0 05 Xylene Cyanol dye pH 8 0 10 mM Tris HCl 0 1 mM EDTA pH 8 0 Solution of N N N N tetramethylethylendiamine 10 Ammonium persulfate 40 glycerol in water 50 glycerol in water 63 17 Subject Index Accessories 52 Buffer Loading buffers 62 Buffers 62 Running buffers 62 Calculate temperatures 35 Declaration of Conformity 57 Decontamination Certificate 54 Disposal 56 DNA Denaturation 24 Renaturation 24 Electrophoresis Setup 31 Elution of DNA 43 Error messages 45 Ethidium bromide staining 43 GC clamps 21 Gel Casting 25 Gel solution 28 Genetic fingerprint 31 Installation 13 Legal Notes 10 Maintenance 51 Mutation analysis 30 Operation Voltage 15 Paralle
27. ON OL SYINPO see en 11 2 2 Safety instructions general remarks nennen ennnnnnneennnnnnnn 11 3 a E UE Le BE 13 3 1 KEE Eege ee 13 3 2 SEH 13 3 3 Unpack and CHEcK EE 14 3 4 instalation CORSO See ee era 14 3 5 Operation Voltage nennen ennnnnnnenen 15 3 6 el eee eae NE EE E E ee ea ee ee ee eee ee EI ee ee eee ener 15 4 Considerations for successful TGGE experiment ccccccsseesssseeeeseeeeeseeesseeeeneeees 16 4 1 Design of TGGE expermente 17 4 2 Design of DNA Tragment for TOGE eu ea 17 4 2 1 Rei ie kan 17 4 2 2 CLORO LLA OSEE 21 4 2 3 Chemical clamp with Psoralen Furo 3 2 g coumarin C1 HgQs 21 4 2 4 Use of SSOP DIME ee ee 22 4 3 Find correct temperature gradient ccccsecccsecceeeceeeecseeeuceceeeceeeseeessueeaeeeseeeees 22 4 4 Parallel analysis of multiple samples 22002240020002200 nano nenn nenn ennne nenne nennen 23 5 Sample PPO al AO IM ee 24 5 1 Bit de e Elei 24 5 2 Sample preparation for direct DNA analves nenne ennnnne nennen nennen 24 5 3 Denaturation Renaturation for heteroduplex analysis of DNA 24 6 CCE e ue acess E ER SEELEN a E 25 6 1 CASHING OL e en ne ee re 25 6 1 1 Assembly of the gel cuvette nenne nenne nennen nenn ennnnnnnnnnnenn 25 6 1 2 Pe Dah OCS OIUNON Eee ee 28 6 2 POUN IS Se E E E ee 29 TGGE Manual October 2009 3 Biometra An Analytik Jena Company 6 3 Electrophoresis with the TGGE System ccccccccc
28. TGGE Manual October 2009 55 Biometra An Analytik Jena Company 13 Note for the disposal of electric electronic waste Note for disposal of electric electronic waste Hinweis fur die Entsorgung von Elektroaltgeraten du traitement des dechets des appareils lectrique lectronique a This symbol the crossed out wheelie bin means that this product should be brought to the return and or separate systems available to end users according to yours country regulations when this product has reached the end of its lifetime Renseignement For details please contact your local distributor This symbol applies only to the countries within the EEA EEA European Economics Area comprising all EU members plus Norway Iceland and Liechtenstein Dieses Symbol die durchgestrichene Abfalltonne bedeutet dass dieses Produkt von der Firma Biometra f r eine kostenlose Entsorgung zur ckgenommen wird Dies gilt nur f r Ger te die innerhalb Deutschlands gekauft worden sind Kontaktieren Sie f r die Entsorgung bitte die Biometra Service Abteilung Au erhalb Deutschlands wenden Sie sich bitte an den lokalen H ndler Dieses Symbol gilt nur in Staaten des EWR EWR Europ ischer Wirtschaftsraum umfasst die EU Mitgliedsstaaten sowie Norwegen Island und Liechtenstein Cet symbol conteneur d chets barr d une croix signifie que le produit en fin de vie doit tre retourn un des syst mes de collecte mis
29. U oOo heteroduplex Aa o Q O homoduplex AA A D homoduplex aa d S warm Figure 22 Schematic drawing of a screening multiple samples in a parallel TGGE Both homoduplices AA aa have a higher melting temperature and migrate further in the gel The heteroduplices melt at a lower temperature resulting in a slower migration 8 5 Evaluation of a heteroduplex analysis There are two possible states in heteroduplex analysis 1 the sample is identical to the standard wildtype 2 the sample is different from the wildtype In the former case the denaturation renaturation procedure results in one the same homoduplex The subsequent separation in parallel TGGE shows only a single band In the latter case denaturation renaturation leads to the four different populations Separation in parallel TGGE therefore results in up to four different bands see 8 6 3 If the temperature gradient has not been correctly optimized or if separation time was to short there may as well only be two or three bands TGGE Manual October 2009 48 Biometra An Analytik Jena Company This makes heteroduplex analysis very easy to evaluate H sample is identical to the standard no mutation more than one up to 4 sample is different form the standard bands mutation 8 6 TGGE Testkit 024 050 The TGGE test kit was developed to get familiar with the TGGE system It contains 3 tubes with different DNA samp
30. ance and allows high parallel sample throughput Using the Biometra TGGE system it is very easy to separate samples either parallel or perpendicular to a temperature gradient All that has to be changed is the position of the buffer tanks Whereas perpendicular TGGE is mainly used for the optimization of separation conditions parallel TGGE allows fast analysis of multiple samples perpendicular TGGE Temperature gradient is perpendicular to the electrophoretic migration One sample is separated over a broad temperature range to determine the optimum temperature gradient or to analyze temperature dependent changes in conformation Parallel TGGE Temperature gradient is parallel to electrophoretic migration multiple samples are separated in parallel TGGE Manual October 2009 7 Biometra An Analytik Jena Company perpendicular TGGE B parallel TGGE cold m 2 77 f if l J cle Oe warm cold u Figure 2 Typical results after perpendicular TGGE A temperature gradient from left to right and parallel TGGE B temperature gradient from top to bottom 1 3 2 Sensitivity Because of the small amount of material used for separation DNA or RNA fragments appear as fine bands which can be clearly distinguished from each other Even complex band patterns can be analyzed due to the high resolution capability of the gradient block Comparing
31. ccsseeeeeeseeeeeeeseeeseeseeeeeeeseaaeees 29 6 3 1 Electrophoresis conditions cccccccceececeeeeeeececeeeecseeeeseeeeseueeseeeesseeeeneeeeseeess 30 6 3 2 Conditions for mutation analysis 30 6 3 3 Conditions for diversity analysis of bacterial populations genetic fingerprint 31 6 3 4 Setup electrophoresis unt 31 6 3 5 Prior to assembly of the electrophoresis um 32 6 3 6 Gel setup for electrophoreSiS nennen nnnnnneennnn 32 6 3 7 Pormendicular TOGE ee ee ee en 33 6 3 8 Parler EE a a a ee Beine 34 6 3 9 Identify optimum temperature gradient nenne nnnnenennennnn 34 6 3 10 Calculate temperatures at different lanes 0 0 eeccceceeeeceeeaeeeeeeseeeeeeeeaaees 35 6 4 Programming the TGGE Controller ccccccsscccceseceeeeeeeeseeeeseeeeeseeeeeseeseetsnaees 37 6 4 1 Software main menu 37 6 4 2 Create edit program 37 6 4 3 Ee EE geeiert 37 6 4 4 Name TOO GAM E 37 6 4 5 Enter temperatures for the gradient block ccccccccceecceeeeseeeeeeeeseeeseeeeseeees 38 6 4 6 Enter electrophoresis D rameterg nenn nenne nenne nennen 38 6 4 7 otal ClO CHOON C EE 39 6 4 8 Stop pause electrophoresis ccceccceceeseceeeeeeececeaeeeeeeseeeeeeeseuueeeseeaeeeessaaeeees 40 6 4 9 View temperatures of the oracdhent nennen 40 6 5 SEENEN ee Seren 41 6 5 1 Print programs E 41 6 5 2 Select de select SiQNal ccccccsssececeeseeeeeseeeeeeeseeeceecseeeeeeseeaeeeeeeeeeeeesaaeeeees 41 6 5 3 CISC WANG MAS ent ee ee
32. d according to their primary sequence there are many potential fields of application For some applications TGGE has become already the TGGE Manual October 2009 29 Biometra An Analytik Jena Company method of choice Currently there are two major fields Mutation analysis in PCR fragments and diversity analysis of complex bacterial samples The main difference between these two applications is that in the former case only one or two PCR fragments mutant and wildtype are separated per lane whereas in the latter case a mixture of many up to 150 fragments are separated in one lane While sample throughput is always an important aspect long separation distance is mainly necessary if many bands are to be separated Therefore the TGGE maxi system is the system of choice for diversity analysis whereas the TGGE standard system allows short electrophoresis times and provides high serial sample throughput for mutation analysis 6 3 1 Electrophoresis conditions The electrophoresis unit of the TGGE System has been designed to accommodate TGGE and all related applications like CTGE TTGE and SSCP without cumbersome changes In contrast to conventional electrophoresis techniques like PAGE or agarose gel electrophoresis the migration behaviour of samples in TGGE depends very strongly on the temperature gradient Better separation in TGGE is NOT achieved by longer electrophoresis times Once the samples start to melt their migration is almost stopped
33. ducibility The most common application for TGGE is mutation analysis of PCR fragments The DNA molecules become separated in the temperature TGGE Manual October 2009 6 Biometra An Analytik Jena Company gradient their melting behavior With TGGE PCR fragments that only have single base substitutions can be resolved But the TGGE system is not restricted to the analysis of DNA fragments only It can be used for versatile applications like Mutation analysis Heteroduplex analysis DNA methylation studies Imprinting Differentiation of amplicon and competitor for quantitative DNA analyses Fidelity assay for thermostable polymerases Secondary structure analysis of RNA Analysis of dsRNA molecules Thermal stability analysis of proteins Protein protein or protein ligand interaction analysis 1 3 Special features 1 3 1 Peltier powered linear temperature gradient The heart of the TGGE system is the temperature block which is powered by peltier technology Thanks to precise microprocessor control a linear temperature gradient is generated providing maximum reproducibility Thus assay conditions can be much better controlled compared to conventional chemical gradients DGGE or temporal gradients using water baths The Biometra TGGE system is available in two formats The standard TGGE mini system operates small gels and is therefore ideally suited for fast serial experiments The TGGE MAXI system provides a large separation dist
34. e gradient 30 to 60 C Voltage 250V mme mm 7 silver stain gel 8 6 3 Gel pictures TGGE Test kit perpendicular TGGE parallel TGGE Te eu TGGE Manual October 2009 50 Biometra An Analytik Jena Company 9 Maintenance and repair 9 1 Cleaning and Maintenance The TGGE system is built to operate for a long time without the need for periodical maintenance Nevertheless occasionally cleaning of the air inlet may be necessary to maintain the efficiency of the thermoblock The inlet for the airflow is located at sides of the themoblock Be sure that the inlet is not clogged by dust or other materials Dust can be removed easily from the inlet with a conventional vacuum cleaner Additionally the housing may be cleaned from time to time with a smooth cotton cloth Do not use strong detergents abrasives or organic solvents for cleaning Important Appropriate safety regulations must be observed when working with infectious or pathogenic material 9 2 Servicing and repair The TGGE system contains no user serviceable parts Do not open the housing instrument Service and repair may only be carried out by the Biometra Service department or otherwise qualified technical personal 9 3 Replacement of Spare Parts Only original spare parts mentioned in these operating instructions are allowed TGGE Manual October 2009 51 Biometra An Analytik Jena Company 10 Accessories 10 1 Consumables and spare parts TGGE Sta
35. een 41 Ze Staining TGGE GGUS E 42 7 1 Silver staining protocol mund 42 1 2 Et idium bromide stainiNg seen nee een 43 7 3 PURO AGORA E 43 7 4 Elution of DNA from the TGGEoel nennen 43 8 Trouble shooting Be e TE 44 8 1 Enor MES AJOS onran AER R 45 8 2 Optimization or paralel E E 46 8 3 Optimization of TGGE for genetic fingerprinting of microorganisms 46 8 4 Optimisation of Heteroduplex analysis nennen nnnnnn nennen nnnnnnn nenne nennen 47 8 5 Evaluation of a heteroduplex analysis u 2uuussnunuenennnenennnnnnnnnnnnennnnn nennen nennen 48 60 TOCE TESKI WANN ee 49 8 6 1 Perpendicular TGGE using the Biometra TGGE test kit nenne 49 8 6 2 Parallel TGGE using the Biometra TGGE Tee 50 8 6 3 Gel pictures TOGE Teerkt nenn 50 9 Maintenance and repair ssssssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnna 51 9 1 Cleaning and Maintenance cccccseeccccsseeeceeeeeceececcegeeeseueeeesueeessageessseeeessaneessaes 51 9 2 SEIVIEING aNd BE 51 993 Replacemento opale Pans serimi nonn ee ee 51 10 PC COS EE ee 52 10 1 Consumables and spare panls 2uerens een 52 TGGE Manual October 2009 4 Biometra An Analytik Jena Company 11 SEENEN 53 11 1 Instructions for return shipment nenne nennn nenne nenn nenne nenne nenne nennen 53 12 Equipment Decontamination Certificate uu u2200002200000nannnn0nnnnnnnnnnnnnnnnnnn 54 13 Note
36. el Buffer wick Buffer wick Figure 15 Setup of gel cover film and buffer wicks 9 Load samples approx 5ul each for parallel gel with 8 slots approx 5Oul for perpendicular gel with 1 slot Note Be careful not to touch the samples with the buffer wick Otherwise the samples will quickly diffuse into the wick 10 Attach gel cover plate the cover plate should have contact to the wicks but should not squeeze gel or wicks 11 Close safety lid and start run Note For parallel TGGE let temperature gradient equilibrate for approx 10 minutes then start main run This step may be omitted for a perpendicular gel 6 3 7 Perpendicular TGGE In perpendicular TGGE one sample is separated over a broad temperature range This application is mainly used to check the melting behaviour of a sample see section 6 3 9 For casting of the gel use a glass plate with one large slot former 024 023 The temperature gradient must be orientated perpendicular to the migration of the sample The buffer tanks must be positioned as described in Figure 16 The migration of DNA RNA molecules is indicated by the arrow on the safety lid of the electrophoresis unit TGGE Manual October 2009 33 Biometra An Analytik Jena Company Gradient o ssarydoao g S G e e 6 Figure 16 Positioning of buffer tanks for perpendicular TGGE Be sure that
37. erature gradient the DNA fragment is separated in a perpendicular TGGE This means the temperature gradient is perpendicular to the migration of samples see section 6 3 7 Thus the migration of a fragment can be checked simultaneously at different temperatures in a single run If the PCR fragment has been designed properly the separation in a perpendicular temperature gradient leads to a distinct melting curve see Figure 7 electrophoresis cold warm T1 T2 Figure 7 Identification of the optimum temperature gradient in a perpendicular TGGE At low temperature below T1 DNA migrates as a double strand left side At intermediate temperature between T1 and T2 the DNA opens at one side the partial double strand is increasingly slowed down Above T2 the DNA separates into the single strands At T1 the double strand starts to melt and forms a branched structure At T2 the partial double strand separates irreversibly into the single strands Analysis of samples in parallel TGGE should be performed precisely in this temperature range between T1 and T2 How to identify the optimum temperature range from a perpendicular gel Place the stained gel on the plastic film with the printed lines I0 to L6 Identify the line where the double strand starts to melt T1 and the line where the double strands separates into the single strands T2 For the calculation of temperature at the corresponding lines see section 6 3 10 TGGE Manual October
38. f the fuse holder and excerpt it Pull out the cream coloured insert from the fuse holder and turn it by 180 Insert it again and control the voltage displayed in the small window of the fuse holder If the setting is correct insert the fuse holder to the power supply again Backside power supply Mains inlet Fuse holder Figure 4 TGGE system fuse holder 3 6 Setup 1 Connect electrophoresis unit and controller 2 Connect power cables from the safety lid to the controller The buffer chambers can be placed in two orientations depending on the direction of the temperature gradient Be sure that the orientation is correct The markings on the gradient block LO to L6 indicate the direction of the temperature gradient The direction of electrophoresis minus to plus for nucleic acids is indicated on the safety lid TGGE Manual October 2009 15 Biometra An Analytik Jena Company 4 Considerations for successful TGGE experiments TGGE is a powerful technique to separate molecules of same size but different sequence Nevertheless every DNA fragment has its own characteristics and three steps have to be taken before successful analysis of multiple samples in parallel TGGE can begin Each of the following steps is described in detail in this section Step 1 Check the sequence of your PCR fragment in Poland analysis The Poland computer program http www biophys uni duesseldorf de POLAND poland html calculates the melting be
39. fer overnight Use a 1 ul aliquot for re amplification TGGE Manual October 2009 43 Biometra An Analytik Jena Company 8 Trouble shooting guide The following trouble shooting guide may be helpful in solving any problem that you may encounter If you need further assistance please do not hesitate to contact your local Biometra distributor or Biometra Problem Cause Solution Electrophoresis Leakage of gel cuvette Inaccurate positioning of Check positioning of silicone sealing sealin g clean Spacer Dust on spacers Acryl glide on spacers do not apply Acryl Glide onto the spacers Acrylamide solution gets Support film is not properly Fix polybond film with adhesive tape behind the support film during attached to the glass plate along the upper edge of the glass pouring the gel plate Teflon film peels away from Block has been cleaned with Contact Biometra the thermoblock strong detergents or aggressive chemicals No current Amperage and Wattage Set Amperage and Wattage to have been set to 0 maximum values Electrophoresis should be controlled by Voltage Current oscillates Coating of the thermoblock Contact Biometra is damaged the safety shutoff is activated Wavelike migration front Temperature inhomogeneity Use as little as possible thermal under the gel due to excess coupling solution not more than 2ml thermal coupling solution No sigmoid melting curve Fragment melts completely Perform Poland analysis
40. for further inquiries with telephone number c Description of the fault which also reveals during which procedures the fault occurred if possible TGGE Manual October 2009 53 Biometra An Analytik Jena Company 12 Equipment Decontamination Certificate To enable us to comply with german law i e 71 StriSchV 817 GefStoffV and 19 ChemG and to avoid exposure to hazardous materials during handling or repair please complete this form prior to the equipment leaving your laboratory COMPANY INSTITUTE ADDRESS PHONE NO FAX NO E MAIL EQUIPMENT Model Serial No If on loan evaluation Start Date Finish Date Hazardous materials used with this equipment Method of cleaning decontamination The equipment has been cleaned and decontaminated NAME POSITION HEAD OF DIV DEP INSTITUTE COMPANY SIGNED DATE PLEASE RETURN THIS FORM TO BIOMETRA GMBH OR YOUR LOCAL BIOMETRA DISTRIBUTOR TOGETHER WITH THE EQUIPMENT TGGE Manual October 2009 54 Biometra An Analytik Jena Company PLEASE ATTACH THIS CERTIFICATE OUTSIDE THE PACKAGING INSTRUMENTS WITHOUT THIS CERTIFICATE ATTACHED WILL BE RETURNED TO SENDER General Information for Decontamination Please contact your responsible health amp safety officer for details Use of radioactive substances Please contact your responsible person for details Use of genetically change organism or parts of those Please contact your responsible person for details
41. for the disposal of electric electronic waste uu2u20022002a00nan0nnnnnnan0nn 56 14 EU Konformit tserkl rung EU Declaration of Conformity uuureennnnnnenn 57 13 NEEN 58 16 PC LE 59 16 1 BEIETERCES EE 59 192 E 62 1621 es toi eier Fu 62 ee Wale UNC E 62 AG SE rel 63 17 SU 0 1 101 4 1 EE 64 TGGE Manual October 2009 Biometra An Analytik Jena Company 1 Introduction Temperature Gradient Gel Electrophoresis is a powerful technique for the separation of nucleic acids or proteins The TGGE method which is covered by patents uses the temperature dependent changes of conformation for separating molecules Since the introduction of the first commercial available TGGE apparatus in 1989 temperature gradient gel electrophoresis has gained high interest in scientific and clinical research laboratories due to the unprecedented resolution capability and easiness of analysis The range of scientific publications using the TGGE method is broad and covers all disciplines which use molecular biology methods e g oncology virology immunology RNA viroid research prion research population analysis The TGGE method has also been used for quantitative analysis in industry and for conformational analysis of proteins 1 1 Principle of the method Conventional protein or nucleic acid electrophoresis separates molecules according to their size or charge TGGE adds a new parameter for separation namely the melt
42. gh quality p a and fresh double distilled water Important Remove the protective plastic sheets from the gel e Carefully remove residual thermal coupling solution from the back of the gel gel support film prior to staining e Put the polyacrylamide gel with the gel side upwards into the staining tray Avoid air bubbles during all staining steps e t s recommended to prepare at least 100 ml solution for each incubation step e Prepare stopping solution prior to developing 7 1 Silver staining protocol ssa Fixation 30 min 100 ml 10 glacial acid 30 EtOH Sensitization 2 x 10 min 100 ml 30 EtOH Washing Rinse gel 30 seconds under running water then wash 5 x 5 min Fresh aqua dest Silver Binding 100ml 0 1 AgNO prepare freshly add 350ul Formaldehyde 37 prior to use Washing Rinse 30 seconds then wash 1 min Fresh aqua dest Rinse again 30 seconds Developing Until bands become visible Solution 1 dissolve 0 2g Sodium thiosulfate Na2S203 in 10ml bidest which can take several minutes Solution 2 dissolve 2 5g Sodium Carbonate Na gt CO in 100ml bidest don t let gel unattended Add 100u solution 1 to solution 2 Add 350ul Formaldehyde 37 TGGE Manual October 2009 42 Biometra An Analytik Jena Company Stopping discard developer Stop solution 100 ml 10 glacial acid add stop solution incubate for 30 min Storage Up to several days at room 10 Glycerol temperature Long term
43. gistration card This is important for you to claim full warranty 3 4 Installation Conditions e Place the TGGE system on a stable surface in a dry safe environment e Let equilibrate the TGGE system to room temperature before starting operation e Make sure that the appliance connector and the plug of the supply cord are accessible SO you Can separate the instrument from the mains e Connect the TGGE system to a grounded socket TGGE Manual October 2009 14 Biometra An Analytik Jena Company Ensure that the ventilation slits at the electrophoresis unit are unobstructed Insufficient ventilation can cause overheating of the instrument Prior to connecting the unit to the power source please ensure that the voltage selector at the back side of the power supply is set to the required voltage Danger of electric shock Unplug the power cable before you open the TGGE system 3 5 Operation Voltage Important Prior to connecting the TGGE system to the mains make sure that the voltage setting of the power supply is in accordance with your mains voltage The set operating voltage is displayed in the small window of the fuse holder at the backside of the power supply The TGGE system can operate at 110 or 230 Volt To change operation voltage of the TGGE system switch off the instrument and disconnect the mains plug Press the two clips both ends o
44. h A list start program 8 Alist Bdel Cquit Denter Confirm program number with D enter The program starts and parameters of gradient block and electrophoresis are displayed During temperature equilibration of the gradient block the elapsed time is displayed Note Electrophoresis starts as soon as the set temperature in the block is achieved LO 25 0 C L6 60 0 C hold 1 2m12s 11 4Vh El 250V 8mA 20 3W A BElpho Cprograms D 6 4 8 Stop pause electrophoresis LO 25 0 C L6 60 0 C hold 1 2m12s 11 4Vh El 250V 8mA 20 3W A BElpho Cprograms D To stop pause the active program press B Elpho program 8 test pause stop A Bpause Cauit Dstop Press B pause to pause program Press D stop to stop program Press C quit to return to the active program 6 4 9 View temperatures of the gradient LO 20 0 C L6 65 0 C hold 1 2m12s 11 4Vh El 150V 8mA 20 3W A BElpho Cprograms D To display the temperatures in the block during a run press A T1 21 1 C T2 63 8 C L133 21 1 32 0 39 0 L3 6 46 0 53 0 60 0 lt const rtime h m The actual temperatures of T1 and T2 as well as the actual temperatures of L1 to L6 are TGGE Manual October 2009 40 Biometra An Analytik Jena Company displayed The limiting factor const V mA or W is indicated by a blinking arrow lt The actual remaining electrophoresis time is shown 6 5 Special functions Main screen LO 20 0 C L6 20 0 C bloc
45. havior of dsDNA molecules Poland analysis can predict whether a fragment is suited for TGGE or not Analysis is available online and free of charge see section 1 5 1 Melting profile is ok Melting profile is not ok Poland analysis shows lf Poland analysis shows that the fragment in its current a satisfying profile state is not suited for TGGE optimize your primer design Never try to separate samples in TGGE if the calculated Proceed with step 2 melting profile is not ok Back to step 1 Step 2 If the Poland analysis shows a suitable melting profile you should test separation conditions in a perpendicular TGGE In perpendicular TGGE a large aliquot of the sample runs over a broad temperature range The result of parallel TGGE allows identification of the temperature gradient for parallel analysis Perpendicular gel is Perpendicular gel is not ok ok If perpendicular analysis does not show the expected Perpendicular TGGE melting profile check sequence again in Poland analysis shows a nice melting Also check purity of chemicals and electrophoretic curve conditions Do not try samples is parallel TGGE as long as the Proceed with step 3 perpendicular gel does not show a defined melting curve Back to step 2 TGGE Manual October 2009 16 Biometra An Analytik Jena Company 4 1 Design of TGGE experiments There are 3 steps in the setup of anew TGGE experiment 1 Design of the PCR fragment 2 Identification of the correct tempe
46. ing behaviour of a molecule The melting behaviour is determined by primary sequence and secondary and tertiary structure of the molecule and can be changed by external influences like temperature salt concentration pH etc During electrophoresis the sample migrates along a temperature gradient As the temperature rises the molecules start to denature Working with PCR fragments for example electrophoresis starts with double stranded molecules At a certain temperature the DNA starts to melt resulting in a fork like structure partial single strand see Figure 1 In this conformation the migration is slowed down compared to a completely double stranded DNA fragment of same size Since the melting temperature strongly depends on the base sequence DNA fragments of same size but different sequence can be separated This is used in mutation detection where PCR fragments of identical size but different sequence are separated Thus TGGE not only separates molecules but gives additional information about melting behaviour and stability cold IUUUUUUUUOUDJD HUTT ds DNA eh partial ss ss DNA electrophoresis warm Figure 1 Different conformations of DNA during temperature gradient gel electrophoresis 1 2 Field of Applications The Biometra TGGE system separates molecules in a temperature gradient Unlike chemical gradients the peltier driven temperature gradient is controlled by a microprocessor and thus is providing unmatched repro
47. iversity analysis of bacterial populations genetic fingerprint For genetic fingerprinting primers against 16SrRNA genes are used Depending on the complexity of the sample and the primer design quite a high number of PCR fragments can arise from one sample In this regard there are two major differences to mutation analysis 1 the number of fragments per lane is much higher 2 the amount of DNA per band is lower To separate all the many fragments a rather flat gradient is used which has to be optimized very carefully For detecting low amounts of DNA a clear staining background is essential The silver staining protocol chapter has been optimized for gels with high content of urea and formamide and thus achieves high sensitivity at low background For microbial diversity analysis we recommend the following conditions However these parameters can only be the starting point for individual optimization Gel composition 1 x TAE buffer 6 Polyacrylamide 37 5 1 8M Urea 2 Glycerol 20 Formamide Temperature gradient L1 33 C L6 44 C Electrophoresis 130V 2h Important Do not denature renature the DNA fragments prior to electrophoresis 6 3 4 Setup electrophoresis unit The Biometra TGGE system is a horizontal electrophoresis system The buffer bridges to the gel are established by layering one side of each buffer wick 024 003 on the gel and submerging the other in the buffer inside the tank To protect the gel from drying it is
48. k off A BElpho Cprograms D Press D to enter the menu for special functions 1 print programs 2 signal 3 language 4 standard mode not occupied 5 test mode not occupied 6 void not occupied An BY Cauit D enter Scroll through the list with Ah BY 6 5 1 Print programs Connect controller to a dot matrix printer Select option 1 in the above menu and confirm with D enter 6 5 2 Select de select signal Select option 2 in the special functions Press A on to activate the signal press B off to inactivate signal 6 5 3 Select language Select option 3 in the special function screen Choose between German and English TGGE Manual October 2009 41 Biometra An Analytik Jena Company 7 Staining TGGE Gels Aside from autoradiography silver staining is the most sensitive method for detecting small amounts of DNA RNA or proteins in polyacrylamide gels Other staining protocols may be used but generally exhibit less sensitivity This must be considered in relation to the amount of DNA loaded on the gel All incubation steps are done in small plastic containers which are agitated on a rocking platform e g order number 042 400 or 042 500 Wear non powdered protective gloves during all steps of the silver staining protocol to avoid staining artifacts due to the high sensitivity of the staining protocol The quality of chemicals is essential in silver staining Prepare solutions fresh use only chemicals of hi
49. k orientation of the buffer tanks parallel or perpendicular TGGE see section 6 3 7 or section 6 3 8 Note Wipe off any spilled buffer from the electrophoresis unit Never run device if buffer has been spilled 3 Soak 2 buffer wicks with running buffer 4 Disassemble gel sandwich Clean backside of the gel support film with a soft tissue 5 Apply 150ul of thermal coupling solution 0 01 Triton on the thermoblock Note The volume of coupling solution should be as small as possible Excess coupling solution leads to an irreproducible temperature distribution under the gel The result is a wavelike migration front and poor separation of fragments 6 Place the gel on the thermoblock The thermal coupling solution should spread over the whole block Avoid bubbles Wipe off any residual coupling solution along the edges of the gel support film Note The thermal coupling solution is essential for efficient heat transfer from block to gel If bubbles are trapped under the gel support film remove support film with gel from the block and place it back again on the block 7 Cover the gel with a cover film The cover film should be placed just beneath the slots 8 Attach pre soaked buffer wicks on top and bottom of the gel TGGE Manual October 2009 32 Biometra An Analytik Jena Company Buffer wick Cover film Cover film G
50. l TGGE Optimisation 46 Poland analysis 17 Program Print program 41 Select language 41 Program create 37 edit 37 enter electrophoresis paramter 38 TGGE Manual October 2009 Biometra An Analytik Jena Company enter temperatures 38 name 37 pause 40 select 37 start 39 stop 40 view temperatures 40 Programming 37 Psoralen 21 References 59 Repair 51 Return shipment 53 Safety and Warning Notices 11 Sample preparation 24 Service 53 Servicing 51 Silver staining 42 Spare Parts 51 SSCP primers 22 Technical specifications 9 Temperature gradient 22 Optimisation 34 TGGE genetic fingerprinting 46 Heteroduplex analysis 47 optimisation 16 Optimisation 17 parallel 7 Parallel 23 34 50 perpendicular 7 22 Perpendicular 33 49 TGGE Applications 6 TGGE Gel Staining 42 TGGE Test kit 49 50 Trouble shooting 44 Warranty 58 64
51. les and 1 tube with loading buffer wild type DNA homoduplex 40 ul Mutant DNA homoduplex 40 ul Mutant wildtype DNA heteroduplex 400 ul Prior to use Samples should to be diluted in 1x TAE running buffer The samples are separated in an 8 PAA gel with 8M Urea and a 1 x TAE buffer system for preparation of gel solution and buffer see section 9 8 6 1 Perpendicular TGGE using the Biometra TGGE test kit 1 sample preparation mix 25 ul heteroduplex sample with 25 ul loading buffer TAE see section 16 2 2 2 Vortex quick spin and load 50 ul in the broad slot of a perpendicular gel 3 Let sample migrate into the gel with 250V for approx 7 minutes 4 Cover gel with cover film assemble buffer wicks cover plate and safety lid 5 start run Temperature gradient 30 to 70 C Voltage 250V eS ee 6 silver stain gel TGGE Manual October 2009 49 Biometra An Analytik Jena Company 8 6 2 Parallel TGGE using the Biometra TGGE Test kit 1 sample preparation mix 2 5ul sample wildtype or mutant or heteroduplex with 2 5ul loading buffer TAE see section 16 2 2 2 Assemble electrophoresis unit cover gel with cover film beneath the slots assemble cover plate and safety lid 3 Vortex quick spin and load 5 ul of wildtype mutant and heteroduplex samples 4 Let sample migrate into the gel with 250V for approx 7 minutes 5 After pre run cover gel like described in section 6 3 4 6 start main run Temperatur
52. nd Baverstock P R 1997 Detecting mitochondrial genotypes by temperature gradient gel electrophoresis and heteroduplex analysis BioTechniques 23 982 986 Poland D 1974 Recursion relation generation of probability profiles for sequence specific macromolecules with long range correlations Biopolymers 13 1859 1871 Lerman L S and Silverstein K 1987 Computational simulation of DNA melting and its application to denaturing gradient gel electrophoresis Meth Enzymol 155 482 501 Steger G 1994 Thermal denaturation of double stranded nucleic acids prediction of temperatures critical for gradient electrophoresis and polymerase chain reaction Schumacher J Randels J W and Riesner D 1983 A two dimensional electrophoretic technique for detection of circular viroids and virusoids Anal Biochem 135 288 295 Sambrook J Fritsch E F and Maniatis T 1989 Molecular cloning Cold Spring Habor Laboratory press Steger G and Riesner D 1992 Temperaturgradienten Gelelektrophorese eine Methode zur Analyse von Konformationsubergangen und Mutationen in Nukleinsauren und Proteinen In Radola B J ed Handbuch der elektrophorese VCH Verlagsgesellschaft Weinheim Sheffield V C Cox D R and Lerman R M 1989 Attachment of a 40 base pair G C rich sequence GC clamp to genomic DNA fragments by the polymerase chain reactiob results in improved detection of single base changes Proc Natl Acad Sci USA 86 232 23
53. nic or radioactive material Ask the responsible local safety inspector for details TGGE Manual October 2009 Biometra An Analytik Jena Company Place the TGGE system on a stable non flammable surface in a dry safe environment Ensure that both the ventilation slits of the TGGE block not clogged by dust or other material Danger of overheating Do not fill buffer chambers above marking for maximum level Do not move instrument during operation In case of strong condensation under the safety lid stop run dry instrument and re start If buffer has been spilled on the electrophoresis unit clean it carefully before start of electrophoresis Do not use paraffin oil or Kerosene on the thermoblock Switch off power before removing the safety lid This instrument is designed and certified to meet EN 61010 1 safety standards It should not be modified or altered in any way Alteration of this instrument will void the warranty void the EN61010 1 certification and create a potential safety hazard Do not use alcohol e g methanol ethanol organic solvents or abrasives to clean the instrument Biometra An Analytik Jena Company 3 Installation 3 1 Content of delivery 1 Electrophoresis unit including thermoblock buffer chambers safety lid 2 Controller incl power supply control of electrophoresis parameters Voltage and temperature gradient 3 Starter Kit Glass plates sealings cover films polybond films
54. not drop repelling solution on the spacers of the glass plate This will lead to leakage of the gel cuvette during polymerization Do not treat the bonding plate with repelling agent Apply some silicone grease onto the spacers to prevent leakage Note Treatment of the glass plate with slot formers should be repeated prior to every use 5 Use original pre cut Polybond support film which perfectly fits onto the gradient block Remove protecting paper sheet from polybond film prior to assembly of sandwich Handle Polybond film only with powder free gloves Attach Polybond film firmly to the bonding glass plate by gently rubbing with a soft tissue To improve contact between glass plate and film a drop of water may be applied behind the Polybond film Note The Polybond support film has a hydrophilic coating The gel matrix binds covalently to the support film In earlier batches Polybond film was coated only on one side Today both sides can be used for gel casting the orientation of the film is not important Note The support film may be fixed along the upper side of the cover glass plate with ordinary adhesive tape This way no gel solution can accidentally get behind the support film TGGE Manual October 2009 26 Biometra An Analytik Jena Company Bonding plate 024 021 Poybond support film 024 030 ee Glass plate with slot fomer and spacers ee 024 022 Figure 10 Assembly
55. of the TGGE cuvette for casting gels 6 Assemble gel sandwich and fix it with the 3 plastic clamps Note the clamps should be placed directly on the spacers 7 Set gel sandwich upright to pour the gel Figure 11 Final setup of the gel sandwich Figure 12 Gel casting stand Note For convenient casting of 5 gels in parallel we recommend gel casting stand 024 028 Figure 12 TGGE Manual October 2009 27 Biometra An Analytik Jena Company 6 1 2 Preparing gel solution The choice of the buffer system has a strong impact on TGGE analysis Concentration of salt and denaturing agents urea or formatted strongly affects the melting temperature of DNA and proteins In general urea is used for the separation of nucleic acids in a concentration between 7 and 8M Urea reduces the melting temperature and thus enables a separation at lower temperatures which is favorable because at higher temperatures the gel tends to dry out To further reduce the melting temperature deionized formamide may be used in concentrations of up to 20 The most popular buffer systems for TGGE are TBE TAE and MOPS In the following two standard protocols for TBE and MOPS buffer systems are listed Please note that the buffer system should be adapted to each special kind of application Each gel sandwich contains approx 2 5 ml polyacrylamide solution We recommend to prepare 10 ml solution to pour 3 4 gels at a time Gels
56. optimal primer sequence may be 5 Pso pTaPpnpnp 3 given the preference of Psoralen for binding between TpA and ApT pairs 13 46 47 Crosslinking of the PCR product is done e g in a flat bottom microtiter plate using a 365 nm UV source Working with small volumes it may be necessary to minimize evaporation by cross linking at 4 10 C The yield is not affected by TGGE Manual October 2009 21 Biometra An Analytik Jena Company temperature The distance of the sample from the UV source affects the yield 15 min at 0 5 cm distance of the sample from an 8 W UV lamp is sufficient 4 2 4 Use of SSCP primers In many cases primer from SSCP may be used for TGGE analysis Nevertheless the resulting DNA fragments should be checked in the Poland analysis If there is only one melting domain add a GC clamp to one of the primers see section 4 2 2 4 3 Find correct temperature gradient Poland analysis gives the first indication which temperature gradient should be applied for parallel analysis of multiple samples Under experimental conditions separation is performed in the presence of high concentrations of urea Urea lowers the melting temperature of the DNA This is important because gel electrophoresis at very high temperatures may lead to partial drying of the gel resulting in a disturbed separation pattern Therefore it is necessary to identify the optimum temperature gradient under experimental conditions To identify the optimum temp
57. r return shipment In case of an instrument failure that cannot be fixed by the procedures described in section 8 please proceed as follows e Return only defective devices For technical problems which are not definitively recognisable as device faults please contact the Technical Service Department at Biometra Tel 49 551 50881 10 12 Fax 49 551 50881 11 e mail service biometra com e Please contact our service department for providing a return authorization number RAN This number has to be applied clearly visible to the outer box Returns without the RAN will be not be accepted e Important Carefully clean all parts of the instrument of biologically dangerous chemical or radioactive contaminants If an instrument is contaminated Biometra will be forced to refuse to accept the device The sender of the repair order will be held liable for possible losses resulting from insufficient decontamination of the device e Please prepare written confirmation that the device is free from biologically dangerous and radioactive contaminants The declaration of decontamination see section 12 must be attached to the outside of the packaging e Use the original packing material If not available contact Biometra or your local distributor e Label the outside of the box with CAUTION SENSITIVE ELECTRONIC INSTRUMENT e Please enclose a note which contains the following a Sender s name and address b Name of a contact person
58. rams can contain different steps pre run pause run Display LCD display 4 lines English German parallel Centronics serial RS 232 Voltage 110 230 V E E l Dimensions DxWxH Weight S TGGE Manual October 2009 9 Biometra An Analytik Jena Company 1 5 Legal Notes 1 5 1 Copyright All rights reserved It is not allowed to copy and publish the manual or parts of it in any form as copies micro film or other methods without a written authorisation from Biometra Biometra is pointing out that applied company and brand names are usually protected trade marks The TGGE method is covered by patents issued to Diagen now QIAGEN GmbH The polymerase chain reaction PCR process is covered by patents issued to Hoffman La Roche Acryl Glide is a trademark of Amresco Inc Biometra is a trademark of Biometra GmbH The POLAND software service established by Gerhard Steger Department of Biophysics University of Duesseldorf is available by internet www biophys uni duesseldorf de POLAND poland html 1 5 2 Liability Biometra is not liable for damages and injuries caused by use not considering these operating instructions in parts or completely 1 5 3 Meaning of the Instructions Biometra recommends that you first read these instructions carefully This operation instruction is part of the product and should be kept over the full life time of the instrument It should also be forwarded to subsequent owners and u
59. rature gradient 3 Parallel analysis of multiple samples 4 2 Design of DNA fragment for TGGE The design of the DNA fragment is an important step for successful TGGE Starting with the gene fragment of interest PCR primers should be designed with a conventional computer program The melting behavior of the resulting fragment should then be checked with the Poland software It is essential that the DNA fragment shows different melting domains If there is only one single melting domain an artificial higher melting domain called GC clamp must be added during PCR 4 2 1 Poland analysis The melting profile of a DNA fragment can be analyzed with a computer program The Poland software calculates the melting behaviour of a DNA fragment according to its base sequence This software is free accessible via the internet http www biophys uni duesseldorf de POLAND poland html How to perform a Poland analysis e Open start page URL see above e 1 enter a name for the query e 2 copy paste DNA sequence in the sequence window e 3 choose the Tm plot de activate all other plots a 4 submit query e 5 retrieve Tm plot melting curve TGGE Manual October 2009 17 Biometra An Analytik Jena Company Poland service request form The Poland server will calculate the thermal denaturation profile of double stranded RNA DNA or RNA DNA hybrids based on sequence input and parameter settings in this form NEW Thermodynamic parameters se
60. rature gradient the DNA fragment is separated in a perpendicular TGGE This means the temperature gradient is perpendicular to the migration direction see section 6 3 7 Thus the migration of one sample can be checked simultaneously at different temperatures in a single run If the PCR fragment has been designed properly the separation in a perpendicular temperature gradient leads to a distinct melting curve see Figure 18 electrophoresis T1 T2 cold warm Figure 18 Identification of the optimum temperature gradient in a perpendicular TGGE At low temperature below T1 DNA migrates as a double strand left side At intermediate temperature between T1 and T2 the DNA opens at one side the partial double strand is increasingly slowed down Above T2 the DNA separates into the single strands At T1 the double strand starts to melt and forms a branched structure At T2 the partial double strand separates irreversibly into the single strands Analysis of samples in parallel TGGE should be performed precisely in this temperature range between T1 and T2 6 3 10 Calculate temperatures at different lanes Place the stained perpendicular gel on the TGGE thermo block showing lanes L1 to L6 Identify at what lane the double strand starts to melt T1 and at what lane the double strands separates into the single strands T2 TGGE Manual October 2009 35 Biometra An Analytik Jena Company g ie IS
61. rter Kit Germany only 024 003 1 glass plate 1 slot 024 023 1 glass plate 8 slots 024 022 1 glass plate 12 slots 024 025 3 bonding plates 024 021 4 electrode wicks 024 016 25 Polybond films 024 030 Acryl Glide sample 211 319 3 plastic clamps 010 007 TGGE Starter Kit International 024 093 1 glass plate 1 slot 024 023 1 glass plate 8 slots 024 022 1 glass plate 12 slots 024 025 3 bonding plates 024 021 4 electrode wicks 024 016 25 Polybond films 024 030 3 plastic clamps 010 007 TGGE buffer wicks 8 x 7 cm 100 pcs TGGE glass plate 9 x 9cm without spacer TGGE glass plate 9 x 9cm 8 slots 5ul 0 5mm spacer 024 022 TGGE glass plate 9 x 9cm 1 slot 50ul 0 5mm spacer 024 023 TGGE cover films 7 x 6 cm 100 pcs TGGE Test Kit 024 050 40ul wildtype DNA 40ul mutant DNA 400ul heteroduplex DNA 1ml loading buffer manual TGGE gel casting stand for 5 gel sandwiches 9 x 9cm 024 028 TGGE Manual October 2009 52 Biometra An Analytik Jena Company 11 Service Should you have any problems with this unit please contact our service department or your local Biometra dealer Biometra GmbH Service Department Rudolf Wissell StraBe 14 16 D 37079 G ttingen Phone 49 0 5 51 50686 10 or 12 Fax 49 0 551 50 686 11 e mail Service biometra com A If you would like to send the unit back to us please read the following return instructions in chapter 11 1 11 1 Instructions fo
62. sandwich to avoid air bubbles Turn gel sandwich into vertical position e Polymerization of gel for 1 1 5 h or over night at room temperature The sandwich should stand up vertically and must not be moved during polymerization Overlaying the gel solution with 2 Butanol will yield an even gel surface after polymerization e After polymerization remove clamps Remove the glass plate without spacers by sliding the glass plate away from the rest of the sandwich if you have fixed the support film with adhesive tape remove or cut tape first The gel must stick on the support film e Slowly take off the Polybond film with the adhering polyacrylamide gel from the other glass plate In the area of the slot former remove the Polybond film very carefully to avoid any damage to the slots e Do only use perfect shaped slots because otherwise the bands will be distorted Note After dissolution of urea batch solution may be stored up to 2 weeks at room temperature Remove clamps and wrap gel sandwich including glass plates in wet paper towels Store the sandwich in a tight plastic bag Gels must not be stored at low temperatures i e refrigerator because this will lead to precipitation of urea Note Prepare electrophoresis unit prior to disassembling the gel cuvette The gel should not be exposed to the air for extended periods since this may lead to drying of the gel 6 3 Electrophoresis with the TGGE System Since in TGGE molecules are separate
63. sers Make sure that additions and updates are inserted into the operation instructions TGGE Manual October 2009 10 Biometra An Analytik Jena Company 2 Safety and Warning Notices 2 1 Definition of Symbols Symbol EFP Definition Caution Refer to instruction manual Danger High voltage Fragile Danger Hot surface 2 2 Safety instructions general remarks SEPP SE gt gt gt TGGE Manual October 2009 The thermoblock will reach high temperatures during operation The thermoblock can burn you The TGGE system contains no user serviceable parts Do not open the housing instrument Service and repair may only be carried out by the Biometra Service department or otherwise qualified technical personal Do not use the instrument when damages of the housing block cable or other parts are visible Prior to connecting the unit to the power source please ensure that the voltage setting at the fuse holder at backside of the power supply is set to the required voltage Unplug the power cable before you open the TGGE system Danger of electric shock The thermoblock is covered with Teflon film Avoid damaging this film Make sure that the appliance connector and the plug of the supply cord are accessible so you can separate the instrument from the mains Connect the TGGE system to a grounded socket Appropriate safety regulations must be observed when working with infectious pathoge
64. setting with D enter 1 L0 25 0 L6 60 0 time 10m Os El OV 500mA 30W A Bdelete C quit D enter Enter Voltage and accept with D enter 1 L0 25 0 L6 60 0 time 10m Os El 250V 500mA 30W A Bdelete C quit D enter Note The values for amperage mA and wattage W are set to maximum level as default If you enter lower values these parameters may become limiting during electrophoresis Accept default settings for voltage and wattage with D enter or enter different values Note We recommend to control electrophoresis by constant voltage rather than by constant amperage set mA to maximum value respectively accept default value Note Each program can consist of several steps Thus it is possible to program complex protocols including a pre run a pause for handling of the gel and the main run In the following screen you can program a second step for your protocol 2 L0 L6 A B CquitD gt lf you do not want to program another step accept program with C quit The program name number of steps and the total run time is displayed program no 8 pgm end 1 step s run time 0h10m Os A B CquitD gt 6 4 7 Start electrophoresis Main screen LO 20 0 C L6 20 0 C block off A BElpho Cprograms D To start a program press B Elpho TGGE Manual October 2009 39 Biometra An Analytik Jena Company start program Alist Bdel Cquit Denter Enter program number or choose a program from the list wit
65. should be used within 24 hours after polymerization To prevent gel drying wrap polymerized gels into saran foil or wet plastic bags Wet paper towels may be used for short time storage 1 day The below gel composition is intended for mutation analysis with the TGGE test Kit 024 050 Gel protocols for other applications can be found in section 6 3 Polyacrylamide is a toxic substance Skin contact or inhalation must be strictly avoided Wearing laboratory gloves is mandatory during all steps Please carefully read the safety sheets of the chemicals prior to use and behave accordingly Protocol for 10 ml gel solution 3 gels Volume Final amount concentration 40 Acrylamide Bis Acrylamide 37 5 1 40 Glycerol 500 ul e Stir solution at lt 50 C until urea is completely dissolved e Carefully degas gel solution e Let solution cool down to room temperature and start polymerization with APS Ti C e Once TEMED and APS have been added the following steps must be performed without delay e Load gel solution in a syringe and attach a 0 4um or 0 25um sterile filter TGGE Manual October 2009 28 Biometra An Analytik Jena Company e Pour gel through sterile filter into the glass sandwich 6 2 Pouring gels Figure 13 Pouring polyacrylamide gel e Hold gel sandwich at an angle of 45 when pouring The solution should run slowly along one side of the plate
66. steep A or a flat B temperature gradient With a smaller temperature gradient 30 to 40 C B the separation of samples is much wider Note the temperatures in this Figure are only for demonstration 8 3 Optimization of TGGE for genetic fingerprinting of microorganisms Primers should be designed thoroughly to avoid unspecific PCR products Ideally sequences of the species considered must be compared for identifying conserved regions Further the PCR cycles should be reduced to a minimum while extension times should be enlarged to avoid the formation of heteroduplex artefacts In cases of mixed template PCR products a low cycler number reamplification may increase specificity In general PCR products should be purified prior to TGGE analysis Sometimes PCR artefacts are co amplified which can result in smears on TGGE gels PCR products can be purified by PCR purification kits or ideally each PCR product is subjected to conventional agarose electrophoresis After TGGE Manual October 2009 46 Biometra An Analytik Jena Company electrophoretic separation for each sample the desired band can be cut from the gel and purified 8 4 Optimisation of Heteroduplex analysis If the difference in melting temperature between wildtype and mutant is very small heteroduplex analysis is a rewarding approach Heteroduplex analysis makes it very easy to distinguish between the wildtype and mutant form of a DNA fragment The basic principle is to mix
67. t for dsDNA in 75 mM NaCl Blake amp Delcourt added Calculation is based on Poland s algorithm in the implementation described by Steger Graphics results are directly sent to your WWW client 1 Sequence title line TGGE Manual October 2009 18 Biometra An Analytik Jena Company Sequence plain format 2 no numbers max 1000 nts min 5 nts Mismatched positions sd comma separated numbers Thermodynamic parameters oe Oligonucleotide Long double strand Dissociation constant B 3 function of seq length default B 1 0E 3 M TGGE Manual October 2009 19 Biometra An Analytik Jena Company e EI Strand concentration Ir default 1 0E 6 M Low temperature limit default 40 0 C High temperature Temperature limit default 110 0 step size Temperature range C default 2 0 C Melting DIM Tn p 50 plot 3d plot Mobility glo H melting 3 Which graphics do you want curve Im u u u Graphics size GIF format submit 4 Click here to sum or Zur cksetzen Click here to he form to defaults The Tm plot second order red color shows the melting profile of the DNA fragment according to the base sequence The ideal fragment shows at least two distinct melting domains Note that mutations can be detected in all but the highest melting domain This means that in a DNA fragment with two melting domains mutations can only be detected in the lower melting domain roland analysis
68. t on the melting behavior because the number of base pairs between the two strands is reduced Therefore the heteroduplices can be easily separated from the homoduplices using TGGE The identification of the optimum temperature gradient for the separation of a heteroduplex analysis is absolutely the same as for a single fragment The separation of a heteroduplex sample in a perpendicular TGGE results in 4 different melting curves The 2 heteroduplices have a lower melting temperature and denature at a lower temperature compared to the homoduplices TGGE Manual October 2009 47 Biometra An Analytik Jena Company heteroduplex Aa heteroduplex aA nn electrophoresis homoduplex aa homoduplex AA cold warm Figure 21 Separation of a heteroduplex sample in perpendicular TGGE The temperature gradient can then be adapted in the same way as for a conventional sample see chapter 6 3 9 In parallel TGGE the samples melt as they migrate along the temperature gradient The heteroduplices with mismatch melt at a lower temperature than the homoduplices Thus they open earlier in the partial single strand and are slowed down in the gel matrix The homoduplices migrate a longer distance as complete double strands and start to melt at a higher temperature i e later in respect to the temperature gradient Therefore the lower bands in parallel TGGE are the homoduplices whereas the higher bands are the heteroduplices cold CITT U
69. the TGGE method with other screening methods like SSCP the superior performance of the TGGE method becomes evident In contrast to direct sequencing TGGE also detects mutations in mixed DNA samples Whenever heterozygous DNA is to be analyzed direct sequencing will not give a clear signal at the position of the mutation This is especially the case if the mutated gene is masked by a high background of normal cells TGGE reliably detects mutations in a 1 10 dilution and higher of wildtype DNA 1 3 3 Patented technology TGGE is protected by patents in most countries of the world The patent for the TGGE method is held by Qiagen AG Hilden Biometra is the exclusive licensee for manufacturing and distribution of TGGE instrumentation TGGE Manual October 2009 8 Biometra An Analytik Jena Company 1 4 Technical specifications TGGE System Electrophoresis unit with temperature gradient block and two removable buffer chambers power supply starter kit Electrophoresis unit Temperature gradient High performance Peltier technology formation Temperature range 5 80 C gradient Parallel 5 cm Sample number volume 10 Gul 12 Gul 18 1 5ul Dimensions DxWxH 23 X 23 X 23 cm TGGE System Microprocessor driven control of temperature gradient and electrophoretic Controller parameters Dimensions DxWxH 31 x 22 x 12 cm Weight 3 8 kg up to 100 programs can be stored Program modes constant Voltage V h integration Prog
70. the direction of electrophoresis is perpendicular to the temperature gradient The temperature gradient is indicated by lines L1 to L6 6 3 8 Parallel TGGE In parallel T GE multiple samples are separated along the temperature gradient For casting of the gel use glass plate with 8 12 or 18 slots 024 022 025 or 026 The buffer tanks for parallel TGGE must be positioned as depicted in Figure 17 Gradient 4 4 2 3 ae Ce 3 Hectrophoresis Figure 17 Positioning of buffer tanks for parallel TGGE Be sure that the direction of electrophoresis is parallel to the temperature gradient The slots of the gel should be at the same side as the markings on the block 6 3 9 Identify optimum temperature gradient Poland analysis gives the first indication which temperature gradient should be applied for parallel analysis of multiple samples In real life electrophoresis is performed in the presence TGGE Manual October 2009 34 Biometra An Analytik Jena Company of high concentrations of denaturing agents urea and Formamide Both lower the melting temperature of the DNA fragment This allows electrophoresis at lower temperatures preventing partial drying effects in the gel resulting in a disturbed separation pattern Therefore it is necessary to identify the optimum temperature gradient under experimental conditions To identify the optimum tempe

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