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1. Microcentrifuge tubes 36 Vortexer optional Micropipettes p10 p200 p1000 Pipette tips PCR tubes 27 Tube Racks Ethanol or ethanol wipes Electrophoresis equipment Electrophoresis supplies agarose TBE buffer DNA loading buffer running buffer gel dye e g SYBR safe Gel Red UV light box or Gel Doc equipment and program Permanent marker GenoSensor DNA Fingerprinting Kit Il GenoSensor Corp Student Guide Objective overview 1 Understand how DNA is responsible for genotypic differences between individuals 2 Investigate techniques used in DNA technology DNA sequence diversity and uniqueness PCR gel electrophoresis 3 Investigate and understand the process for gel electrophoresis including analyzing band pattern data In this lab you will examine an abridged version of a DNA analysis PCR During the exercise you will learn to analyze and compare a number of DNA fragments to determine whether or not they are from the same individual These fragments can be visualized through a process known as gel electrophoresis DNA is long double helix polymer that uses deoxyribose rings Sugars and phosphate molecules as support in its backbone Attached to the backbone are unique sequences of nucleotides which are often referred to as base pairs There are two different types of nucleotides purines and pyrimidines Adenine A and Guanine G are both purines because they have two rings in their structure2. cheek cells etc After proper treatment with PCR millions of copies of nearly any desired DNA sequence can be produced The power of PCR is its specificity PCR uses unique primers to target just the desired sequence of DNA out of the entire genome and amplifies only that segment with little error The basic components of PCR Reaction Buffer DNA nucleotides dNTP s of adenine guanine thymine and cytosine DNA polymerase Forward and reverse DNA oligonucleotide primers Template DNA starting material PCR Makes Use of Two Basic Processes in Molecular Genetics 1 Complementary DNA strand hybridization For DNA to be amplified one must have a known sequence that flanks the gene of interest both upstream and downstream These sequences are used to create oligonucleotide GenoSensor DNA Fingerprinting Kit Il GenoSensor Corp 11 primers meaning a short 20 base pair nucleotide sequence which is used as a starting point for DNA replication The primers are complementary to their target regions so they will anneal attach to those regions with great precision Primers serve the same purpose that runways do for planes trying to lift off the ground DNA polymerase cannot add nucleotides without a preexisting chain to start from This process is referred to as primer extension DNA polymerase recognizes the partially single stranded segment of DNA attaches itself to the primer just as it normally would during the DNA replication
3. Light sensitive dyes should be preparation kept in the dark during gel preparation Prepare in dark room or place a box over the electrophoresis apparatus during gelation and electrophoresis Expired contaminated or degraded Verify that the DNA dye has DNA dye not degraded in storage been contaminated or expired Non specific Premature Taq polymerase Mix solutions on ice place rxn amplification replication directly into 94 thermal cycler product Primer annealing temperature too Raise annealing temperature low in 22C increments Insufficient mixing of reaction solution Mix solutions thoroughly before beginning the reaction Exogenous DNA contamination Wear gloves Use dedicated area for sample preparation Use non aerosol tips GenoSensor DNA Fingerprinting Kit Il GenoSensor Corp Technical Service For more information or technical assistance please call write fax or email GenoSensor Corporation 4665 S Ash Avenue Suite G 18 Tempe Arizona 85282 Tel 1 480 598 5378 Fax 1 480 755 3319 Email tech_service genosensorcorp com Web www genosensorcorp com Limited Warranty GenoSensor is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about a GenoSensor product or service please contact our Technical Service at tech_s
4. phase of the cell cycle and proceeds to add complementary nucleotides to fill in the gap Complementary strand hybridization occurs when two different oligonucleotide primers anneal to each of their respective complementary base pair sequences on the template They are designed specifically to anneal at opposite ends of opposite strands of the specific sequence of DNA that is desired to be amplified 2 DNA strand extension via DNA polymerase In a PCR a special type of DNA polymerase is used that is able to function properly and not become denatured during the temperature fluctuation cycles required for thermal cycling Most mammalian DNA polymerases cannot tolerate the high temperatures and fluctuations from 60 C 94 C The breakthrough in PCR came with the isolation of DNA polymerase from a thermophilic bacterium known as Thermus aquaticus This bacterial species lives in high temperature steam vents and its DNA polymerase has evolved to withstand the high temperatures of its environment During PCR DNA is synthesized and its quantity doubles after each cycle making the reaction product grow at an exponential rate In theory after 30 cycles there will be 2 over a billion copies of DNA Yielding this much DNA allows it to be visualized after only a few simple procedures One of the easiest and most popular methods of doing this is agarose gel electrophoresis Genes and DNA The human genome contains 23 pairs of chromosomes that contai
5. Background Introduction to PCR In 1983 during his time at Cetus Corporation Kary Mullis developed a technique that significantly changed the field of genetics and that of all other biological sciences This revolutionary process was termed polymerase chain reaction or PCR By 1993 he had earned the Nobel Prize in Chemistry for PCR His new technique enabled researchers in numerous fields of biology to easily and rapidly amplify DNA Before that amplification of DNA was extremely difficult and time consuming Now in the 21st century it s not just research scientists who use this molecular biology technique PCR has applications in a wide variety of areas including gene detection and mapping whole genome sequencing analysis of gene expression forensics criminal justice clinical diagnostics pharmacogenomics and dozens of others Nearly every one of these applications were impossible prior to the implementation of PCR Besides the initial investment in specialized machinery the cost of performing PCR is relatively low and the process is simple enough that nearly anyone can do it and get successful results every time PCR The Birth of Recombinant DNA Technology PCR uses specific nucleotide sequences named primers to amplify segments of a genome from a very small amount of starting material referred to as the template DNA can be extracted and isolated from almost any cell type i e bacterium blood cells tissue cells hair cells
6. G GenoSensor Corporation eee GenoSensor DNA Fingerprinting Kit II Catalog 4002 Version A July 2015 User s Manual GenoSensor DNA Fingerprinting Kit II Manual Table of Contents Notes Tor INSTRUCTORS onn cei ue ier eerta o rhe raa deae be tevaeenaewaveicaveewmecswlenehs 2 Shipping Storage and Safety eeeeeeeeeeeeeee 3 GenoSensor DNA Fingerprinting Kit Il Overview 4 Kit Components and Storage Conditions c ccccccccsssssssscecececessessnneseeeeeeeeseseeaeeeceesseeseasaeeeeeeeseeseaeaeesesens 4 Additional Required Materials ccccccccsssssscccccessssesnsaecececessesesaeseeeeeceseeeaaeaeeeescesseseeaeseeeeseesseseaaeaeeeesees 4 jueripgtnIdqe EL 5 Full iioii el ht 7 FADS CULLING 2t erento ttem deni i e tmu sumeret ad m A eT EE Rer eas 7 Reagent PreparatiOn a aeter cte delete ea bere ead sate cet Reve tae Pee a HER ge gs ptr qua eee vx aet 7 PCR Reaction inp REI aa eene p auteni e 7 Agarose Gel Electrophoresis rn en rr er I E re e EE a ERRORS 8 Results and DISCUSSIOn net eeu aenea ie cee dieaeden ceececeaueecdecbedeceCusdivaedeetevaceceuswecseddubscetesdweedeedeneees 10 GenoSensor DNA Fingerprinting Kit Il Background 11 Troubleshooling eene eeepc ere noun Ce eae acu 16 Technical SerVICG 5 5curoxuvdugexituzCesuva cau rano eevo uv du Gisldep
7. If the teacher did not pre aliquot samples add the DNA samples THEN the master mix enzyme to your microcentrifuge tubes changing tips each time Store the samples on ice until they are ready to be loaded into the thermal cycler PCR Parameters Program the thermal cycler as follows 1 arwn 6 94 C 30 seconds 94 C denaturing 20 seconds 58 C annealing 20 seconds repeat steps 2 3 amp 4 for 35 cycles 68 C extension 30 seconds 68 C 5 minutes 4 C finished hold STOPPING POINT For classes with shorter time periods the PCR samples should be stored at 4 C until the next lab period Agarose Gel Electrophoresis General Procedure detailed directions as given by instructor 1 2 Prepare 196 agarose Set up electrophoresis apparatus and pour in the 1 molten agarose with DNA dye for gelation For staining use a DNA dye which is added directly to the molten agarose For light sensitive dyes keep the gel in the dark during gelation either by performing in a dark room or placing a box over the gel Use at least 10 uL of PCR product to visualize results by electrophoresis on agarose gel If gel well volume will accommodate more than 10 uL a higher volume is preferred Loading dye has already been added to the sample to ensure that the sample will sink to the bottom of the well and properly enter the agarose gel Run at 100V for 20 minutes and stop before loading dye has run off gel Dep
8. PCR on Page 2 Pre Experiment Observations 1 Describe the samples of DNA physical properties color viscosity etc Can you see the DNA 2 Is there any observable difference between the samples of DNA 3 Describe the appearance of the 2X PCR Master Mix Can you see the enzymes PCR Reaction Keep the 2X PCR Master Mix and all samples on ice when not in use 1 Wear gloves and handle solutions carefully Spin the MM tube for 10sec vortex for 10sec then spin for another 10sec 2 Using a NEW TIP for each sample pipette 10 uL of the 2X PCR Master Mix containing Taq DNA polymerase nucleotides primers and PCR reaction buffer into the sample tubes A B C and U already containing 10 uL of each DNA and the N tube if your team received one PCR Reaction Mixtures DNA Samples 2X PCR Master Mix Total Reaction Volume Unknown DNA U 10 uL 10 uL 20 uL Sample A A 10 uL 10 uL 20 uL Sample B B 10 uL 10 uL 20 uL Sample C C 10 uL 10 uL 20 uL Negative control N 10 uL 10 uL 20 uL 3 reactions 3 Pipette up and down carefully to mix well Tightly cap each tube Alternatively mix the components by gently flicking the tubes with your finger Arrange the tubes in a microcentrifuge and spin for 5 seconds to force all liquid to the bottom of the tubes Be sure the tubes are in a BALANCED arrangement in the rotor GenoSensor DNA Fingerprinting Kit Il GenoSensor Corp 4 NOTE
9. e eGesirut cade a bao eW E HEP CERE 17 Literature Citation When describing a procedure for publication using these products please refer to them as the GenoSensor DNA Fingerprinting Kit Il GenoSensor DNA Fingerprinting Kit Il GenoSensor Corp Notes for Instructors Kit Components and Storage Conditions Component Storage 2X PCR Master Mix 20 C Sample A 20 C Sample B 20 C Sample C 20 C Unknown DNA 20 C Negative control 20 C DNA ladder 20 C Preparation for PCR for 6 teams Set up thermal cycler and the PCR program Thaw 2X PCR Master Mix on ice Spin Master Mix for 10 seconds then vortex for 10 seconds spin for another 10 seconds Label 6 microcentrifuge tubes MM and aliquot 40 uL of 2X PCR Master Mix into three tubes and 50 uL into the other three tubes KEEP ON ICE Label 6 tubes 24 total each A B C and U and aliquot 10 uL of each DNA sample store on ice Label 3 tubes N and aliquot 10uL of negative control store on ice In class distribute 1 each MM A B C U tubes to all teams and 1 N tube to three teams they will share later for electrophoresis Be sure to give the 50 uL MM tubes to the teams that also have the N tube Students will use 10 uL of 2X PCR Master Mix with 10 uL sample DNA for a final PCR volume of 20 uL Electrophoresis Electrophoresis reagents are not provided in the kit Please refer to the Additional Required Mate
10. e mixed as a 2X PCR Master Mix in the GenoSensor DNA Fingerprinting kit l The tubes are placed into the thermal cycler which contains an aluminum block that holds the samples and can be rapidly heated and cooled by extreme temperature changes in a controlled environment The rapid heating and cooling of this thermal block is called temperature cycling or thermal cycling The first step of the PCR temperature cycling procedure heats the sample to 94 C causing the template strands separate This process is called denaturation The thermal cycler then rapidly cools to 60 C allowing the primers to anneal to the separated template strands This is the annealing process The two original template strands may re anneal to each other or compete with the primers at the primers complementary binding sites However the primers are added in excess so that the primers may out compete the original DNA strands for their complementary binding sites Lastly the thermal cycler heats the sample to 72 C the usual environment temperature for Thermus aquaticus so that Taq DNA polymerase can perform primer extension and produce complementary DNA strands of the target sequence The two resulting new sets of GenoSensor DNA Fingerprinting Kit Il GenoSensor Corp 13 double stranded DNA dsDNA will be used for the next cycle and proceeding strand synthesis At this stage a full temperature cycle thermal cycle will have been completed Each step takes 30 seconds t
11. ending on the DNA dye used caution may need to be taken to reduce exposure of gel to light Visualize under UV light exposure and record the results manually or by photography Suggested Gel Setup Run 3 gels with 10 wells 2 teams gel Team 1 samples A B C and U up Lane 1 4 i520 uL each Lane 5 10 uL DNA Ladder GenoSensor DNA Fingerprinting Kit Il GenoSensor Corp Lane 6 10 uL Negative Control Team 2 samples A B C and U up Lane pelos 20 uL each GenoSensor DNA Fingerprinting Kit Il GenoSensor Corp Base Pairs Mass ng 1 517 45 1 200 35 1 000 95 900 27 800 700 21 600 18 500 517 Fig 1 100 bp DNA Ladder Results and Discussion Observe the bands visible in your samples on the gel Recall which lanes contained the samples and which contained the unknown DNA sample Do any of the samples match the unknown DNA sample on the gel Looking at the bands in relation to one another is quick and useful What would be a more accurate way to infer band size and their distance traveled Compare the results from your gel with those of the other teams Describe the similarities and differences Summarize the process of PCR using the correct terminology Describe a new experiment you could perform using PCR and DNA agarose gel electrophoresis GenoSensor DNA Fingerprinting Kit Il GenoSensor Corp 10 GenoSensor DNA Fingerprinting Kit Il
12. ervice genosensorcorp com GenoSensor warrants that all of its products will perform according to the specifications stated on the certificate of analysis This warranty limits GenoSensor Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions GenoSensor reserves the right to select the method s used to analyze a product unless GenoSensor agrees to a specified method in writing prior to acceptance of the order GenoSensor makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore GenoSensor makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Service GenoSensor assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose GenoSensor DNA Fingerprinting Kit II GenoSensor Corp
13. he Linnaean system uses seven different tiers of classification in order to properly name every species because the genetic diversity on earth is so great The differences between these genomes underlie the theory behind DNA profiling There are a number of specific regions in our genomes that vary reliably between individuals For this experiment the focus will be ona Variable Number Tandem Repeat VNTR region Throughout the genome there are segments that feature small repeating sequences of DNA A repeated sequence is generally the same GenoSensor DNA Fingerprinting Kit Il GenoSensor Corp between individuals but the number of times it repeats can vary By analyzing enough of the VNTR segments a genetic fingerprint for an individual can be generated Genomes contain many random insertions by short repetitive interspersed elements SINEs and long repetitive interspersed elements LINEs Those elements have become randomly inserted within our genome mostly in introns over millions of years VNTR and Alu elements are the most common Polymerase Chain Reaction PCR technology is a powerful tool to examine and compare genetic variations GenoSensor DNA Fingerprinting Kit Il GenoSensor Corp Full Protocol Lab Setting Materials are enough for 6 groups Preparation 1 Setup and program the thermal see below 2 Thaw 2x PCR Master Mix on ice Reagent Preparation Refer to Notes for Instructors Preparation for
14. in protein coding genes arise the result is very evident and is sometime catastrophic Many metabolic disorders and rare diseases are caused by mutated and nonfunctional proteins Introns however often vary in size and number among individuals Intron sequences are thought to be the result of the differential accumulation of mutations over time and through evolution are silently passed on to descendants after reproduction It is differences in the presence and number of intron sequences that allow us to determine the diversity of human genetics The recognition of these distinctive characteristics in DNA represents the molecular basis from which human identification and population genetics are made possible Throughout evolution intron sequences have been the target of random insertions by short repetitive interspersed elements SINEs or long repetitive interspersed elements LINEs Those elements have become randomly inserted within our introns over millions of years PCR Stages The machinery required to perform PCR is known as a thermal cycler The thermal cycler enables the steps of PCR to be automated The reaction involves a repetition of cycles that promote template denaturation primer annealing and primer extension by the Taq DNA polymerase A DNA sample is added to a mixture of the necessary reagents oligonucleotide primers thermostable DNA polymerase Taq the four nucleotides A T G C and reaction buffer These reagents are pr
15. n a total of thirty to fifty thousand protein coding genes However those genes only comprise about 5 of the genome leaving 95 of it to be classified as non coding DNA This non coding DNA is found not only between but within genes splitting them into segments In eukaryotes non coding DNA sequences found within genes are known as introns The sequences that do code for proteins are called exons In eukaryotes genomic DNA is transcribed into RNA molecules in its unmodified form containing both introns and exons from a particular gene While the RNA is still in the nucleus before being transported out of the nucleus the introns which interfere with the gene product must be removed from the RNA while the exons excised from the original transcript are spliced together to form the complete messenger RNA sequence which will soon be translated into a protein This process is called RNA splicing Some genes may contain a few GenoSensor DNA Fingerprinting Kit II GenoSensor Corp 12 introns others may contain dozens Interestingly it is the non coding junk DNA that is useful to us when considering the DNA profile of an individual rather than the protein coding DNA previously thought to be the most aspect of the genome As discussed functional segments of genes exons code for proteins Proteins are molecules that carry out most cellular functions Exon sequences are therefore very similar among individuals That is why when mutations
16. n or eyes if contact should occur wash immediately with water and follow your laboratory safety protocols Safety Data Sheets for products are available upon request GenoSensor DNA Fingerprinting Kit Il GenoSensor Corp GenoSensor DNA Fingerprinting Kit Il Overview The GenoSensor DNA Fingerprinting Kit II introduces common techniques used in DNA research and in forensic analysis The kit creates a crime scene scenario utilizing three different plasmids to represent three suspect samples labeled Sample A B and C one of which matches the Unknown sample representing criminal DNA collected from the scene of the crime The goal of the experiment is to identify which of the suspects is the culprit by performing a polymerase chain reaction PCR on the four samples After completing the experiment students will be able to understand the concepts behind PCR gel electrophoresis and the genetic concepts driving the experiment Kit Components and Storage Conditions Materials for 6 teams Component Amount 27 rxns Storage 2X PCR Master Mix 270 uL 20 C Sample A 60 uL 6 rxns 20 C Sample B 60 uL 6 rxns 20 C Sample C 60 uL 6 rxns 20 C Unknown DNA 60 uL 6 rxns 20 C Negative control 30 uL 20 C DNA ladder 30 uL 20 C Additional Required Materials Thermal Cycler Heat Block or heat plate Beaker with de ionized water water bath Tube floater Thermometer Ice Microcentrifuge
17. o 1 minute and will repeat for 30 40 cycles depending on how the user has programmed the thermal cycler At the end of the pre programmed number of cycles the product is put on hold at 4 C until the user is ready to retrieve the PCR product and analyze its contents GenoSensor DNA Fingerprinting Kit Il GenoSensor Corp Genomic DNA Extraction Genomic DNA m Primers Anneal and Polymerase Extents PCR Amplification ean Gel logie zh mr am onl a en Figure 3 Experiment flowchart from start to finish GenoSensor DNA Fingerprinting Kit II GenoSensor Corp Troubleshooting Symptom Possible causes Solutions No amplification product Questionable template quality Analyze starting material Inhibitory Substance in reaction Decrease sample volume Insufficient cycle Run additional cycles Incorrect thermal cycler program Verify times and temperatures Errors in heat block incubation Calibrate heating block use sand or water to maximize contact with tube for proper heat transfer Contaminated tubes solutions Autoclave tubes and use filter tips Primer annealing temperature too high Lower annealing temperature in 2 C increments Weak bands faint Low concentration of DNA template Make sure enough DNA has signal been added see recommended amount of DNA to load into PCR reaction DNA Dye degradation during
18. rials list on page 4 Best results are obtained by adding DNA dye i e Gel Red Sybr Safe to molten agarose For light sensitive DNA dyes avoid exposing the agarose gel to light It is best to store and run the gel in a dark room or cover the gel with a box during gel polymerization and the whole electrophoresis process DNA ladder supplied is enough for 3 lanes with 10 uL each Negative control supplied is enough for 6 lanes with 10 uL added after PCR DNA samples A B C and U supplied is enough for up to 20 uL lane for 6 teams GenoSensor DNA Fingerprinting Kit Il GenoSensor Corp Shipping Storage and Safety Shipping and Storage GenoSensor DNA Fingerprinting kits are shipped on blue ice Components should be stored at temperatures shown in the above table At proper storage conditions components are stable for 1 year from the date received Expiration dates are also noted on product labels Safety Warnings and Precautions This product is intended for research use only It is not recommended or intended for the diagnosis of disease in humans or animals Do not use internally or externally in humans or animals Consider all chemicals as potentially hazardous Only persons trained in laboratory techniques and familiar with the principles of good laboratory practice should handle these products Wear suitable protective clothing such as laboratory coats safety glasses and gloves Exercise caution to avoid contact with ski
19. s Meanwhile Thymine T and Cytosine C are pyrimidines because they have only a single ring in each of their structures These nucleotides form a bond with their complementary base pair on the other strand of DNA This is how the double helix structure is formed that resembles a spiral staircase Each individual will have different sequences of A T G and C in their DNA There are highly similar and yet unique sequences of DNA that are used to identify humans by looking at the minute differences in their DNA In this exercise you will use several techniques to figure out if the DNA in any of the three samples matches up with the DNA of the unknown sample In this exercise you will use several techniques to figure out if the DNA in any of the three samples matches up with the unknown DNA sample We are very different from each other in many ways but not as much as you might think on the genetic level Our genome consists of over 3 billion base pairs and yet the genetic makeup from one person to the next may differ by as little as 0 1 Evidently that 0 1 still makes a huge difference Organisms need to be able to differentiate their species from that of closely related species increasing genetic diversity is what makes that possible for them Genetic diversity is the driving force behind speciation in any population of organisms In the 1700 s Swedish scientist Carl Linnaeus devised his hierarchical classification system for naming organisms T
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