Lab #4 Polymerase Chain Reaction

Transcription

1 BE60, 011 Lab #4 Polymerase Chain Reaction In this laboratory work you will perform an allelespecific PCR. Allelic variants mean that the two genes (two alleles) are present in one DNA sample. These alleles originate from maternal and paternal DNA. If the maternal and paternal genes are identical and do not have mutations it is a wild type. If one of the genes is mutant we have a heterozygous phenotype; if both genes have the same mutation we have heterozygous mutant phenotype. You will amplify a DNA fragment of the human gene encoding Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). You will study the most often mutation, F08 in the CFTR gene, which causes a devastating disease, cystic fibrosis. The F08 mutation consists in absence of three nucleotides in the CFTR gene. CF is a recessive disease which means that both alleles should have the mutation for the person to be sick. Your goal is to estimate the genotype of the DNA sample. Each student will receive a DNA sample containing a fragment of human genomic DNA. This DNA fragment was preamplified from individual human genomic DNA that might or might not contain mutation F08 in the CFTR gene. PCR should reveal the presence or absence of the wild and mutant alleles. The PCR products will be analyzed by gel electrophoresis in the agarose gel. Based on the PCR results you will estimate the genotype of the CFTR locus (wild, mutant heterozygous or mutant homozygous) in your DNA sample. Lab skills: polymerase chain reaction (PCR), allelespecific PCR, DNA agarose gelelectrophoresis, UVdetection of DNA Equipment: Thermocycler, apparatus for gel electrophoresis, UVtransilluminator, gel electrophoresis documentation Tasks: 1. To learn how to design PCR primers. To perform PCR 3. To analyze PCR products using agarose gel electrophoresis 4. Estimate the CFTR genotype of genomic DNA based on PCR results Lab report: In the Introduction briefly describe the PCR method. Make a note about the allelespecific PCR. You may also include a brief description of the major mutation F08 leading to cystic fibrosis In the Materials and Methods briefly describe the PCR components and conditions of amplification. Describe the DNA sample as a blind test, a DNA fragment preamplified from human genomic DNA at CFTR locus with the two genespecific primers. Describe also gel electrophoresis to analyze the results. In the Results and Discussion focus on the results of amplification. Incorporate the gel picture in your report. 1

2 Introduction The polymerase chain reaction (PCR) is a technique for exponential amplification a DNA fragment via enzymatic replication in a test tube. PCR can be used for amplification of a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. As PCR is an in vitro technique, it can be performed without restrictions on the form of DNA, and it can be extensively modified to perform a wide array of genetic manipulations (Wikipedia). General PCR scheme Each cycle results in duplication of target DNA molecules, so that after n cycles their number will increase n times if the efficiency were 100%. DNA synthesis is performed by a thermostable DNA polymerase, which survives C denaturing steps and has maximum activity at 77 0 C. PCR conditions and yields Typical cycling conditions Step 1: DNA denaturation Step : Primer annealing Step 3: Primer extension C for 3060 sec 6 0 C for 30 sec 7 0 C for 60 sec (go to step times)

3 and so on. Usual key component concentrations: Target Primer dntps Mg + DNA polymerase <10 1 M (10 9 and down to as little as 1 molecule) x M x 10 8 M of each 1 mm Allelespecific PCR of CFTR gene Three possible DNA genotypes DNA genotypes WILD MUTANT HETEROZYGOUS MUTANT HOMOZYGOUS, the mutation Note that in this Figure each line denotes double stranded DNA and two lines correspond to two DNAs (one from the mother and another from the father, in other words two DNA alleles) Genomic sequence of a fragment of the CFTR gene 961 tttacaaata agaatataca cttctgctta ggatgataat tggaggcaag tgaatcctga 101 gcgtgatttg ataatgacct aataatgatg ggttttattt ccagacttca cttctaatga 1081 tgattatggg agaactggag ccttcagagg gtaaaattaa gcacagtgga agaatttcat 1141 tctgttctca gttttcctgg attatgcctg gcaccattaa agaaaatatc atctttggtg 101 tttcctatga tgaatataga tacagaagcg tcatcaaagc atgccaacta gaagaggtaa 161 gaaactatgt gaaaactttt tgattatgca tatgaaccct tcacactacc caaattatat 131 atttggctcc atattcaatc ggttagtcta catatattta tgtttcctct atgggtaagc 3

4 F08 deletion (ctt) is marked by yellow; the direct and reverse primers used to preamplify CFTR fragment are underlined. Find the positions of the wild and mutant primers and calculate the length of the expected allelespecific PCR product in case of the wild and mutant genotype. Expected PCR results for three different allelic variants wild mutant homozygous heterozygous M M M Allelespecific primers: 1,, no DNA controls with two sets of primers 3, DNA + primer set for the wild allele 4, DNA + primer set for the mutant allele M, PCR markers CFTR_dir_W4 gcaccattaa agaaaatatc atct (note the allelediscriminating nucleotides) CFTR_dir_M4 gcaccattaa agaaaatatc attg Common primer: CFTR_rev gggtagtgtg aagggttcat atgc 4

5 To reveal DNA CFTR genotype you will run two allelespecific reactions and use two sets of primers: the common reverse primer will be combined either with the CFTR_dir_W4 or with CFTR_dir_M4. Thus, everybody will run two allelespecific PCRs and two nodna controls and analyze the PCR products on the agarose gel. Laboratory work (please read carefully) 1. Prepare ice bath, take out all stock solutions (PCR buffer, dntps, MgCl, primers and DNA polymerase and DNA) and put them into ice.. Table 1 shows you the volumes of the PCR components for the µl final reaction volume 3. Based on the volumes given in Table 1, prepare master mix for reactions (you need to run 4 reactions: two allelespecific PCRs, plus two nodna controls, thus if you do it fold you will have some excess of the master mix). Master mix contains all the reaction components except for DNA and primers. Use 1. ml Eppendorf tube for master mix preparation. This is an example of the master mix preparation (final volume of the PCR reaction is µl, use 1. ml Eppendorf tube) Mix for 1 tube for tubes (x 1, µl) (x, µl) 10 xpcr buffer. 1.. mm dntps. 1. mm MgCl. 1. Taq DNA polymerase 0. µl/tube 0.. H O 1 60 Total Aliquote µl of the master mix in 4 small PCR tubes, add µl of DNA to the tubes 1 and, then add µl of the primers marked wild in tube 1, primers marked mutant in tube. Add µl of water to tubes 3 and 4 (instead of DNA) and wild and mutant primers, correspondingly. Mix and spin down the mixture. Table 1. PCR setup ## tube 1 3 (no DNA control) 4 (no DNA control) Deioniz ed water PCR master mix DNA (3 ng/µl) Primer mix wild,. µm each CFTRrev+dirW4 Primer mix mut,. µm each CFTRrev+dirM4 Total volume

6 . Put the tubes into PCR machine and start thermocycling. Thermocycling program (B1 on the head A): Step 1: 94 0 C, min, Step : 94 0 C, 10 sec, Step 3: 0 C, 1 sec Step 4: 7 0 C, 30 sec (go to step times). 6. While PCR is running, prepare % agarose gel in 1 x TAE buffer, add ethidium bromide after agarose has melted (1 µl/100 ml). One gel can be used by two students. 7. Mix on the piece of parafilm µl of the dye and 10 µl to the PCR product. 8. Load all 1 µl on the gel; load µl of 100 bp size markers on the side (New England Biolabs, catalog number #N331S) 9. Run the gel 10. Take picture in the UVlight using Polaroid handhold camera and Polaroid film Example of the results after agarose gel electrophoresis: In this experiment two DNA samples were probed in allelespecific PCR (no controls without DNA are shown) The conclusion from this experiment is that DNA 1 was amplified only with the normal (wild) allele primer and was not amplified with the mutant primer. It means that this DNA is homozygous normal or wild. DNA was amplified only with the mutant primer and was not amplified by the normal primer. The conclusion is that DNA is homozygous mutant ( F08). DNA 1 DNA Composition of the buffers and of the loading dye 1. 1 x TAE buffer: 40 mm Trisacetate, 1 mm EDTA, ph x PCR buffer: 10 mm TrisHCl, ph 8.3, 0 mm KCl 3. 6 x Loading dye (contains bromphenol blue, xylene cyanol, and orange G): 0% sucrose, each dye concentration is <1%. Additional reading: Chapter 4 in Cantor& Smith Genomics 6