PROJECT 2:

PCR-Based Cloning to Detect Polymorphisms in a Human Gene

 

OBJECTIVES

             

            This lab will use the polymerase chain reaction (PCR) to isolate part of a gene, CYP2D6, from the student’s DNA.  CYP2D6 encodes a cytochrome P450 enzyme that is responsible for the metabolic breakdown and processing of many different drugs, including codeine and Prozac.  The DNA sequence obtained with PCR will be analyzed and compared to published polymorphism databases to determine the effects of any detected polymorphisms on the student’s gene coding potential and phenotype.  The analysis will illustrate how reverse genetics and sequence analysis can be used to correlate a sequence polymorphism to a specific phenotype.

 

INTRODUCTION

            When scientists sequence an organism’s genome, they obtain large amounts of sequence information about various genes, but usually have no idea what these various genes do or why they are important.  Reverse genetics allows researchers to study this genotype information by placing mutations in the gene sequence and studying what effect(s) (if any) those mutations have upon normal phenotype.  However, in certain model organisms, like humans, these types of genetic manipulations cannot be performed for a variety of ethical reasons.  Thus, as researchers try to make sense of the Human Genome Project’s sequence information, they must use the genetic variations already present in humans to study the effects of polymorphisms on normal gene function and phenotype.

            One example of how researchers use genetic variations to study human genes is in the study of human disorders.  To study which polymorphisms may cause a disorder and how they may do so, the researcher must first collect a large group of people, some of whom have the disorder and some of whom are (or appear to be) healthy.  The researcher then sequences the gene(s) suspected to be involved in that disorder in all of his/her research subjects.  The researcher analyzes this sequence information and must try to find a particular polymorphism that is more commonly associated with the disease than with healthy people (or vice versa).  This approach uses the genotype information (sequence data) obtained from the research subjects to determine or predict the phenotype (disease state) of a person.

            The gene that we are studying in this lab, CYP2D6, is responsible for the detoxification of certain chemicals in the body.  Previous studies have linked various polymorphisms in this gene to differing abilities by some people to take certain drugs.  These polymorphisms have been neatly compiled in a table, accessible at http://www.imm.ki.se/CYPalleles/cyp2D6.htm.  We will use this published data to compare your CYP2D6 gene sequence to these known polymorphisms.  If there is a match, it may indicate that you have an altered phenotype and may need to use caution when taking certain drugs (but consult your doctor first!)  Thus, our goal in this lab is to use a reverse genetics approach to extract genotype information about you (your CYP2D6 gene sequence) and use it to predict your phenotype (your ability to take certain drugs).  This phenotype data will be kept private between yourself and your instructor; however, if you feel uncomfortable collecting this information or do not wish to analyze your own DNA, talk to your instructor to make alternate arrangements for this lab. 

BACKGROUND INFORMATION

Cytochrome P450 genes

            Cytochrome P450 monoxygenases are a class of enzymes that uses electrons originating from NADH to oxidize certain substrate molecules.  Like other cytochromes, they are membrane proteins; however, they function independent of the electron transport chain of respiration and are usually located in the endoplasmic reticulum rather than in the mitochondria.  Most eukaryotes possess multiple genes, termed CYP genes, which encode different cytochrome P450 enzymes, each of which (usually) has its own specific substrate molecule(s).  The CYP genes determine the rate of stage I detoxification of drugs and compounds encountered in our diet or environment. 

            Humans possess 115 CYP genes, 57 of which are functional and 58 of which appear to be pseudogenes.  These genes are divided into four gene families (CYP1, CYP2, CYP3, and CYP4) by sequence similarity; for example, all CYP1 genes are more closely related to each other than to CYP2 genes.  Further sub-grouping within each family follows added letters and numbers (e.g. CYP1A1). 

            Cytochrome P450 enzymes interact with drugs and chemicals in a variety of ways as they process these compounds into forms that can be excreted by the body.  In the process of metabolizing a drug, these enzymes may activate a compound (like the conversion of the inactive drug codeine to its active form, morphine) or deactivate a compound (like the conversion of dextromethorphan to its metabolite dextrorphan).  The processing of a drug can have harmful side effects, however; the aflatoxins found in Aspergillus-infected corn or peanuts become mutagenic only after being processed and activated in the liver.  The liver damage caused by some herbal supplements could also result from similar activation.

The normal function of cytochrome P450 enzymes can be altered by other drugs or compounds encountered in food or dietary supplements.  Some can induce the expression of the CYP gene, while others can serve as enzyme inhibitors.  For example, compounds in grapefruit juice affect drug metabolism by the CYP3A4 gene, and taking vitamin E while on Prozac will decrease the breakdown of Prozac.  Thus, there is significant interaction between the chemical environment and the activity and expression of different CYP genes.
            In addition to environmental influences, genetic variation among individuals can alter the activities of cytochrome P450 enzymes.  As mentioned before, polymorphisms in CYP genes have been linked to differences in the ability to breakdown and process drugs.  The polymorphisms implicated in these differences are both large-scale (duplications, deletions and recombinations between different CYP genes) and localized (point mutations that alter either the promoter sequence or the coding sequence).  The distribution of the polymorphisms has been correlated to gene flow in the human population over time, to cancer susceptibility, and to drug susceptibility.  Since we will be looking at ethically neutral phenotype information (drug sensitivities as opposed to life expectancies or behavioral predispositions), we are most interested in those correlations that link specific polymorphisms to varying levels of drug detoxification activity.  

CYP2D6 gene

            The CYP2D6 gene is located on chromosome 22, and is flanked on one side by two pseudogenes--CYP2D7 CYP2D8.  The enzyme encoded by CYP2D6 is named debrisoquine hydroxylase, so-named because debrisoquine is one substrate of the enzyme.  However, the enzyme also acts on a number of at least 30 other compounds, including codeine (conversion to morphine), opiate derivatives, dextromethorphan  (an ingredient in many over-the-counter cough medications), Ritonivar (an anti-HIV protease inhibitor), Prozac, vitamin E, MDMA (“Ecstasy,” (+/-)-3,4-methylenedioxymethamphetamine), and simvastatin (a cholesterol-reducing drug). 

            Measuring the amount of enzyme activity in an individual can give a good idea as to  how well that person is able to metabolize certain drugs.  Enzyme levels that vary from expected levels can indicate an enzyme deficiency, and drug dosage may need to be adjusted accordingly.  One way to measure enzyme activity in vivo is to measure the amount of metabolic product present in the body, either in the blood stream or in urine.  More accurately, one could measure the ratio of the original compound to its metabolic by-product.  For example, one could determine debrisoquine hydroxylase activity by measuring the urine ratio of dextromethorphan to its hydroxylation product dextrorphan. 

            Measurements such as these have revealed a wide range in CYP2D6 enzyme activity in the human population.  Several groups of enzyme activity levels stand out; in particular, poor metabolizers (PM), intermediate metabolizers (IM), extensive metabolizers (EM) and ultrarapid metabolizers (UM).  The consequences of the extreme groups can be significant.  For example, a poor metabolizer will not clear a drug from the blood in the expected time; thus, a standard dose of that drug would essentially be an overdose and will likely cause harmful or toxic side effects.  Conversely, an ultrarapid metabolizer would clear the drug more rapidly, and a “standard” dose of a drug would not reach the blood concentrations needed over time for the drug to be effective.  These differences in enzyme activity levels cannot be easily detected unless the metabolic ratio of the drug to its by-product was measured in the body.

The genotypes associated with these different phenotypes have been determined by comparing the rates of drug clearance to CYP gene sequence.  When examining the CYP2D6 gene in particular, “UM” individuals have duplicated copies of the CYP2D6 gene so that there are 2, 3,4, 5, or up to 13 tandem copies of this gene.  This duplication event is referred to as the CYP2D6*2 allele.  The “PM” phenotype is associated with several mutated alleles, including CYP2D6*3 (a deletion of A at bp 2637), CYP2D6*4 (a GàA mutation at bp 1934), and CYP2D6*5 (deletion of the entire CYP2D6 gene).  With sufficient numbers of individuals tested to determine these correlations, the presence of a specific genotype can be used to predict the rate of drug metabolism that someone might have and, if needed, plan drug therapies and dosages accordingly. 

The polymerase chain reaction (PCR)

            The polymerase chain reaction is a means of replicating DNA in vitro using the naturally occurring enzyme DNA polymerase.  DNA polymerase requires a 3’ -OH to synthesize new DNA on a template; as such, it cannot just land on a single-stranded DNA to start synthesizing.  Thus, for this reaction to proceed there needs to be a DNA primer—a short, single-stranded DNA oligonucleotide of approximately 15-22 bp that hybridizes to a complementary region on a single-stranded DNA template to “prime” DNA synthesis from the free 3’ -OH of the oligonucleotide.

                                          

   5’  ........TTAGCTGGTAATTTACGGAGTTA.......  3’                  Single-stranded DNA template

                   ||||||||||||||

               3’  GACCATTAAATGCC  5’                 Primer

(Direction of new DNA synthesis)                            

            In PCR, two primers are needed that flank the region of the template DNA you wish to amplify.  The upstream primer corresponds to a known DNA sequence on the top strand (5’ à 3’) of the DNA, the downstream primer corresponds to a known DNA sequence on the bottom strand (3’ à 5’) of the DNA.

 

                   (Upstream primer)

    5’  CTTTGGACTATCT

5’ ...AGCTTTGGACTATCTA.......................CGTAATCTTCTACTTCATC... 3’

3’ ...TCGAAACCTGATAGAT.......................GCATTAGAAGATGAAGTAG... 5’

                                                TTAGAAGATGAAGT 5’   

                                                                                                              (Downstream primer)

We will be using the following two pairs of primers in our PCR reaction:

            4Flong (upstream):               5’  GCC TTT GTG CCG CCT TCG CCA ACC ACT  3’

            2Rlong (downstream):                5’  CCC TCG GCC CCT GCA CTG TTT CCC AGA T  3’

            dupF (upstream):                      5’  CCT GGG AAG GCC CCA TGG AAG  3’

            dupR (downstream):           5’  CAT TTA CGG CAG TGG TCA GCT  3’

                 

                The polymerase chain reaction uses cycles of temperature changes to (1) denature the template DNA (since you need single-stranded DNA as the template), (2) hybridize the primers onto the template, and (3) allow the DNA polymerase to synthesize the DNA.  Since the denaturing step (1) normally requires 94°C, most DNA polymerases would be killed by the high temperature.  To get around this, the DNA polymerase used in this reaction has been isolated from a heat-resistant bacteria (Thermophillus aquaticus); this “Taq polymerase” will not be destroyed by the high denaturation temperature.  The temperature of step (2) is determined by the sequence of the primers used in the reaction.  Step (3) has a temperature around 72°C, the optimal temperature for Taq polymerase activity.  The cycles of steps 1, 2, and 3 are repeated up to 35 times.  The main product of the reaction is a fragment of DNA that corresponds to the portion of the template DNA between and including the primers.  An animation of the PCR reaction can be found at http://allserv.rug.ac.be/~avierstr/principles/pcrani.html.

 

Web resources:

·        Good outline of the CYP2D6 gene, including its alleles:

o       http://herkules.oulu.fi/isbn9514258649/html/x246.html

·        Great overview of CYP genes and drug interactions:

o       http://www.anaesthetist.com/physiol/basics/metabol/cyp/cyp.htm#alpha

o       http://www.nature.com/tpj/journal/v5/n1/pdf/6500285a.pdf  This is the one handed out in class

·        Drug/CYP gene interactions by drug brand name, from a company that genotypes alleles:

o       http://www.pgxlab.com/clin_med.htm

·        1999 review paper about CYP gene polymorphisms:

o       http://www.acb.org.uk/AnnClinBiochem/annals_pdf/Nov99/acb722.pdf

·        Paper linking metabolism with the different alleles and PCR-based allele tests

·                    http://www.aapsj.org/articles/ps0204/ps020433/ps020433.pdf  

·        Analysis of CYP2D6 gene polymorphisms in mainland China:

o       http://www.clinchem.org/cgi/content/full/48/7/983 


OVERVIEW OF LAB PROCEDURES

 

PCR 1:  Collection of Cheek Cells

             

Each student will take a swab of their own cheek to collect cheek (buccal) cells.  The DNA in these cells will be isolated and used for PCR in subsequent labs.  The detergents in the FTA cards will lyse the cells and the DNA will bind the cellulose paper.  Pathogens in the sample will be similarly lysed and, thus, disabled.  If you have concerns about testing your own DNA, talk with the instructor and we can arrange for you to use an anonymous DNA sample.

 

Read Instructions:  Collection of buccal cell samples onto FTA card

 

Web resources:

·        Whatman FTA card description and uses:

·        http://www.whatman.com/References/FTA%20CardsDataSheet%20FINAL%203.25.10%20LR.pdf

·        Whatman FTA card protocol for obtaining buccal cell sample:

o       http://www.whatman.com/References/BUCCAL%281%29.pdf

·        Cross contamination from punch? Experiment with punching a clean spot to test cross contamination

o       http://www.whatman.com/References/Cross%20Contamination%20App%20Note%20FINAL%204.6.10%20LR.pdf

·        General protocol to extract DNA from anything—why would we not use this approach?            http://learn.genetics.utah.edu/content/labs/extraction/howto/


PCR 2:  Processing DNA sample

The FTA card containing the buccal cells will be cut to produce a sample disk.  This disk is then washed during this class period to remove components that may interfere with the PCR reaction.

 

Read Instructions:  Processing of FTA samples

Web resources:

·        Whatman FTA card data sheets and protocols:

http://www.whatman.com/References/BUCCAL%281%29.pdf 

 

PCR 3:  PCR reaction

The prepared disk with the attached genomic DNA will be added to a mix of primers, nucleotides, and Taq polymerase for amplification.  Based upon the specific primers used (4Flong and 2Rlong), a portion of the CYP2D6 gene will be amplified from the genomic DNA.  Since the entire gene is 8 kb in length, it is difficult to amplify the whole gene with Taq polymerase.  It is possible to amplify the entire gene with long-range PCR, but these reactions are more quirky and expensive.  If you have a deleted CYP2D6 gene, you will not get a PCR product—but if you get no product, do not assume you have this gene deletion because it could merely be a problem with the PCR reaction itself.

            We will amplify a 1.2Kb region of the CYP2D6 gene. The first attempt will be to do this directly. A backup will be to amplify a longer 2Kb product, and then amplify the 1.2Kb product in a second PCR.

To detect duplicated copies of the CYP2D6 gene, we will set up a second PCR reaction to detect a duplicated gene.  This reaction will use the dupF and dupR primers that face away from each other (as opposed to facing each other, like 4Flong and 2Rlong do) so that a 3.5 kB PCR product will only result if there are multiple copies of the CYP2D6 gene.  

Read Instructions:  PCR reaction

Questions to Consider:

·        We are using genomic DNA as the template.  How might this PCR product be different if we were to use mRNA or cDNA as the template?

·        What mistakes would you expect if the primers only hybridize partially to genomic DNA?

 

Web Resources:

PCR 4:  Electrophoresis of PCR products

The DNA products from the PCR reaction will be visualized by electrophoresis on agarose gels.  Because the carcinogen ethidium bromide will be added to the agarose gel to stain the DNA, gloves MUST be worn while handling the gel or electrophoresis buffer.  A size standard will be used to measure the size of the DNA band(s) and determine if one of them is the expected PCR product.  We expect a 1.2 kb band from the CYP2D6 exon primers.  If the gene has been duplicated, we expect a 5 kb band from the duplication primers.  The DNA band at approximately 1.2 kb will be cut from the gel and weighed.

 

Read Instructions:  Gel Electrophoresis

Analysis:

  • What is the length (bp) of your DNA?
    • Measure the distance traveled by the molecular weight standards from the base of the well to each the band in the standard.
    • Using semilog paper or a graphing program like Excel, plot the distance traveled from the well (x-axis) vs. the log of the size of the DNA band (in bp) (y-axis).  Connect the plots using a straight (linear) line.
    • Measure the distance the PCR product’s band has traveled from the base of the well.
    • Using the graph created from the molecular weights, estimate the size of your PCR product. 
      • Remember, the y-axis uses a logarithmic scale, so you will have to raise 10 to the power of your result (10x) to estimate the bp size of your product.

 

  • How many different DNA products did your PCR reaction create?
    • Would running the gels for a longer amount of time change your answer?

 

 

PCR5:  Extraction of DNA product from the gel

The gel band containing the PCR product was cut from the gel in the last lab.  In this lab, Quiagen gel extraction kits will be used to extract the PCR product from the gel’s agarose matrix.  The amount of DNA and its concentration after purification by the kit will be determined using a spectrophotometer to measure the solution’s absorbance at 260nm.  We will submit this purified DNA for sequencing; about 900-1000 bases of sequence will come back from the sequencing facility (from our original 1.2 kb product), approximately 800 bases of which will be reliable sequence.

 

Read Instructions:  Quiagen Gel Extraction

Web Resources:  

·        Quiagen Min-Elute description:

o       http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/minelutegelextractionkit.aspx#Tabs=t1

                   manual: http://www.qiagen.com/literature/render.aspx?id=160

 

PCR6:  DNA Sequence analysis
            The DNA sequence obtained from the sequencing facility is returned in two forms.  In one, the chromatograph of the dye elution profiles as detected by the sequencing machine is shown; this is the raw data.  A DNA sequence is displayed above the chromatograph; this is a computer analysis of the bases based on the height and spacing between the peaks in dye intensity.  A second file contains just the analyzed sequence as text.  The text file will be used for the first level of sequence analysis.  However, be aware that sequencing anomalies occur and the computer can misdiagnose the sequence.  The chromatogram file will be the reference for determining the reliability of the sequence and analyzing any mixture of sequences in your PCR product that would result from heterogeneity.

Read Instructions:  Sequence Analysis

             

Web Resources:

·        Look up the known polymorphisms in the CYP2D6 allele:

o       http://www.imm.ki.se/CYPalleles/ 

·        NCBI SNP database for CYP2D6

o       http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?chooseRs=all&go=Go&locusId=1565

·        Gene summary in the Online Mendelian Inheritance in Man database—has history, describes alleles and pharmacological effects:

o       http://www.ncbi.nlm.nih.gov/omim/124030

·        Gene Card for CYP2D6 Weismann Institute

o       http://www.genecards.org/cgi-bin/carddisp.pl?gene=CYP2D6&search=cyp2d6

·        Entrez gene record for CYP2D6

o       http://www.ncbi.nlm.nih.gov/gene/1565

             

 

Additional Analysis of the CYP2D6 alleles in your samples:

            A. Testing for a specific SNP found in CYP2D6*10 allele using PCR and restriction analysis

                        A common SNP in certain Asian and Pacific Islander populations is a C to T transition at the DNA position 100 (relative to the ATG start codon), which produces a missense mutation of proline at the 34th postion in the protein to serine (denoted P34S). This is outside the region we will be sequencing. In order to determine the sequence at this position, we will PCR amplify this region using primers that flank this SNP. Then we will use a restriction enzyme to cut the PCR product at a site that overlaps this polymorphism. HphI recognizes the sequence GGTGA (top strand) or TCACC (bottom strand) and cuts the DNA 7 bases downstream. The T version of this SNP has a HphI recognition site  (TCACC- underlined is the proline codon), while the C version does not have this restriction site (CCACC-underlined is the serine codon) and so is not cut. We will PCR amplify this region, cut the PCR product with HphI, and use gel electrophoresis to determine if your amplified DNA is cut or not. Both alleles can be followed- having one cut and one uncut band indicates heterozygosity at this SNP.

 

            B. Testing for copy number variation (CNV) at the CYP2D6 locus.

                        A variation in the CYP2D6 gene is in the number of copies of the gene one possesses at that gene locus. Some individuals carry duplication of the CYP2D6 gene up to 13 copies at the same location. We will use PCR to detect such duplication. Primers are selected such that they are only positioned close enough to produce a 3.5Kb PCR product if the sample has tandem duplications of the CYP2D6 gene.

DETAILED LAB PROCEDURES

 

PCR 1—Buccal cell sampling

1.      Label your FTA card with your name and date, being careful not to touch the pink area on the card.

2.      Remove the foam tipped applicator from its package.  Place the foam tip in your mouth, and using one of the flat sides of the applicator rub the inside of one cheek for 30 seconds.  Next, rub the inside of the opposite cheek using the other flat side of the foam tip.  Finally, run the foam tip along the gum line and under the tongue, soaking up as much saliva as possible. 

3.      Lift the cover of the FTA card to expose the pink sample area.  Press a flat side of the foam tip to the card within the circle on the card.  Without lifting the applicator, squeeze the tip to the card by rocking the tip gently from side to side three times to saturate the card.  Turn the applicator over and repeat using the other side of the foam tip.  The pink card should turn white in the areas where your sample has been applied.

·        Note:  Try not to grind the foam tip into the card too hard, since the card can be easily damaged when wet.

4.      Discard the applicator in the autoclave bag provided.  Do not reinsert the applicator in your mouth once it has touched the card.

5.      Let the sample dry between lab periods.  To help ensure proper drying, use the paper flap that normally covers the sample area to prop the sample up so that air can reach both sides of the sample.

 

PCR 2—Preparing buccal cell samples for PCR reaction

Punching sample disks

1.      Place the FTA card on the cutting mat with the sample side facing up.  Make sure that other parts of the card are not beneath the sample.

2.      Place the 2 mm Harris MicroPunch coring tool on a white area of the sample.  Do not press on the ejection plunger at this time.

3.      Press firmly down with the coring tool with a slight twisting motion to cut out a circle.

4.      Place the coring tool (with the sample disk in the corer) over a PCR tube and press the ejection plunger.  Repeat this so that you have two disks in the tube—one tube for each reaction. You can wash up to about 12 disks in one 1.5 ml microcentrifuge tube.

5.      Clean the tip by dipping it in ethanol and wiping dry with a Kimwipe.

 Washing disks

1.      Add 200 microliters (¼L) of FTA purification reagent to the tub with the sample disks. If you are washing many disks, use 1 ml.

2.      Incubate the disks in the reagent for 5 minutes at room temperature, occasionally inverting the tube several times every minute or so to make sure the disks are being washed.

3.      Remove the reagent with a pipettor and discard in the waste beaker provided.

4.      Repeat steps 1-3 twice more, for a total of three washes with FTA purification reagent.

5.      Add 200 ¼L of  Tris-EDTA (TE) buffer (10 mM Tris, pH 8.0, and 0.1 mM EDTA).

6.      Incubate disks in TE buffer for 5 minutes at room temperature with occasional mixing as above in step 2.

7.      Remove the buffer with a pipettor and discard in waste beaker.

8.      Repeat steps 5-7 once more, for a total of two TE buffer washes.

9.      Try to move the disks near the top of the tube, and allow to dry at room temperature for 1 hour.  If needed, disks can be dried faster by placing at 56°C for 10 minutes.

·        Note:  The disks should be white after washing.  If they are not, you may need to repeat the washing procedure.

PCR 3—PCR reaction to amplify part of CYP2D6 gene

CYP2D6 Exon PCR (Used in Sequencing)—Ingredients and final concentrations:

1 sample disks, washed

            1X Taq polymerase buffer—contains Tris, detergents, and KCl

            100 microM dNTPs  (dATP, dCTP, dGTP, dTTP)

5 picomoles of each primer  (4Flong and 2Rlong)

            2 units of Taq polymerase

1.0 mM MgCl2  (Mg concentration can range between 1.0 – 3.5 mM, but 1.0-1.5 mM has been found to be optimal for this reaction)

            Distilled water to bring the final volume to 50 microL

1.      Using the following solutions and amounts, set up a PCR reaction to amplify part of the CYP2D6 gene.  Mix these components together in a PCR tube in the order given below.

                 2       washed sample disks

            36.6 microL  distilled water

                                      5.0 microL  10 X Taq polymerase buffer

                                      1.0 microL  dNTP mix

                                      1.0 microL  40 picomole/microL solution of 4Flong primer

                                      1.0 microL  40 picomole/microL solution of 2Rlong primer

                                      0.4 microL  Taq DNA polymerase

2.      Place the PCR tube in the PCR reaction machine.  Let the machine heat the solution to 94°C for the first denaturation step, then add 5 microL of 10X MgCl2 to the tube to start the reaction.  The reaction will be run using the following conditions:

                        Step 1:  Fully denatures genomic DNA in the sample disk 

              94°C for 3 minutes

Step 2:  35 repeating cycles of denaturation, primer annealing, and
                                    polymerase extension

                                        94°C for 45 seconds

                                        69°C for 1 minutes     (temperature uniquely determined for primers)

                72°C for 1.5 minutes  (length of step determined by size of PCR product)

                        Step 3:  Final extension step to ensure reaction is complete

                                        72° for 7 minutes

Nested PCR- a back-up in case some students do not get bands

                        PCR#1:  primers    Ex6F2  5’-AAGAAGTCGCTGGAGCAGTGGGTGA

                                    Ex11Rev 5’-ACCGATGACAGGTTGGTGATGAGTGT

                                    1mM MgCl2

                          

              94°C for 3 minutes; 35 repeating cycles of  94°C for 45 seconds

                                        64°C for 1 minute, 72°C for 2 minutes; Final extension 72°C for 7 minutes

            Take 1 microliter from this PCR reaction, and use it in a PCR reaction using the 4Flong   2Rlong primers as used before, but with no discs added.

 

CYP2D6 Duplicated Gene PCR—Ingredients and final concentrations:

2 sample disks, washed

            1X long-range PCR  buffer-Expand buffer 2- includes MgCl2 to 2.75mM

            350 microM dNTPs

15 picomoles of each primer  (dupF and dupR)

            4 units of Taq polymerase

            Distilled water to bring the final volume to 50 microL

1.      Set up a PCR reaction as above, using the same ingredients (changing the primers to dupF and dupR for this reaction). 

2.      After the PCR machine has heated the reaction mixture to 94°C in the first denaturation step, add Taq polymerase.   Since the product is longer than 3Kb, extra extension time will be added for a long-range PCR reaction. The reaction cycle used for this reaction will be.

  94°C for 2 minutes; 10 repeating cycles of  94°C for 10 seconds, 58°C for 30 seconds, 68°C for 4 minutes; then 25 repeating cycles of 94°C for 10 seconds, 58°C for 30 seconds, 68°C for 4 min 20 sec extending to 12 min 20 sec by the last cycle; a final extension  at 72°C for 7 minutes.       

 

PCR 4—Running an agarose gel to separate PCR products

1.      A 1% agarose gel will be pre-made for you by melting agarose in electrophoresis buffer (a tris-acetate buffer with EDTA) and allowing the agarose-buffer mixture to cool and solidify in an electrophoresis tray.  Ethidium bromide is added to the agar while in the molten state to allow visualization of DNA bands in the gel.  Because ethidium bromide is a carcinogen, you MUST wear gloves when handling the gel.

2.      Take 20 microL of the PCR reaction mixture from last lab and place on a small piece of Parafilm or into a small microcentrifuge tube. 

3.      Add 2 microL of loading dye (60% sucrose with bromophenol blue dye) to the PCR reaction solution and mix together. 

·        The dye acts as an electrophoresis marker, and the sucrose makes the solution sink into the wells of the agarose gel.

4.      Load the sample-dye mix carefully into one well of the gel.  A size marker will be loaded to serve as a reference for the size of your PCR product.

5.      Once all samples are loaded, attach the leads from the power supply to the gel running tray.  Since DNA has a negative net charge, it will move from the negative (black) electrode at the top of the gel (nearest the wells) to the positive (red) electrode at the bottom of the gel.  The gel will be run with a current of 100 volts.

6.      When the bromophenol blue dye has moved about 12 cm, turn off the power supply and bring the tray to the handheld UV light.  This is a low intensity light that will fluoresce the ethidium bromide dye in the gel in the presence of DNA, but will not be so intense as to damage the DNA (which would affect the sequence quality you obtain from your PCR product).  The TA will help you pick out the correct PCR band in your lane of the gel and will help you cut out that band from the gel using a scalpel.  Be sure to handle the gel ONLY with gloves. 

7.      Place the gel slice with your PCR product band in a microcentrifuge tube.  Weigh the tube before and after adding the gel slice to estimate the weight of the gel slice.

 

PCR 5—Removing PCR product from gel and preparing for sequencing

For extraction of the PCR product from the agarose gel slice, we will use a gel extraction kit from Qiagen.

1.      Add 3 volumes of buffer QG (yellow solution) to the tube with your gel slice.  The weight of your slice determines how much of buffer QG to add: 1 mg of gel slice weight  = 1 microliter (microL)). 

·        Example:  If your gel slice weighs 70 mg, you would add 210 microL of buffer QG.

2.      Incubate the gel slice at 50°C to melt the agarose.  Mix the solution every minute to help dissolve the agar. 

·        Watch out for any color change from yellow to purple.  If this occurs, add more QG buffer because the pH of the solution has become alkaline and you need it to be acidic in order to bind the DNA to the gel extraction column (see below).

3.      When the agarose has completely dissolved, add 1 gel slice volume of isopropanol and mix.

·        Example:  If your gel slice weighs 70 mg, you would add 70 microL of isopropanol.

4.      Load the whole solution onto the top tube of a Quiagen column (purple plastic).  Spin for 1 minute in a tabletop centrifuge.

5.      Discard the flow-through liquid in the bottom (clear) tube and add 0.5 mL of QG buffer to the top (purple) tube.  Spin for 1 minute.

6.      Discard the flow-through liquid in the bottom tube and add 0.7 mL of PE buffer to the top tube.  Let the tube sit for about 5 minutes, then and spin for 1 minute.  Discard the flow-thru in the bottom tube.

7.      Spin the empty tube and column again for 1 minute to eliminate any remaining drops of PE buffer in the column.

8.      Move the top purple tube into a new outer centrifuge tube.  Add 12 microL of EB buffer to the center of the purple column (the white disk).  Let it sit for a minute or two before spinning for 1 minute.

9.      This time, keep the small amount of flow-thru in the bottom tube—this is your DNA sample. Discard the top purple tube.

10.   Measure the absorbance of the DNA solution with a spectrophotometer at 260 nm to calculate the DNA concentration.

Sequence Analysis: Click here for Analysis Page –best viewed in Microsoft Explorer

Same Sequence analysis as Adobe Acrobat file: http://www.clfs.umd.edu/classroom/bsci415/straney/lab/PCRanalysis.pdf