Lab 8: PCR (Polymerase Chain Reaction)

PCR – Polymerase Chain Reaction has many applications PCR is commonly used to produce many copies of a selected gene segment or locus of DNA. http://www.slic2.wsu.edu:82/hurlbert/micro101/images/101PCR%20animation.gif

PCR – Polymerase Chain Reaction has many applications In criminal forensics, PCR is used to amplify DNA evidence from small samples that may have been left at a crime scene.

PCR – Polymerase Chain Reaction has many applications PCR can be used to amplify DNA for genetic disease screening or paternity tests.

Lab 8: Obtaining DNA Sample Add cheek cells to Chelex Boil (lyse cells and destroy nuclease) Centrifuge Extract DNA sample

How Does PCR Work? Virtual PCR: http://learn.genetics.utah.edu/ To imbed video into ppt: http://www.insidecancer.org/ PCR Video http://teachercenter.insidecancer.org/p/989 (mrsschramm@yahoo.com)

Marty’s PCR Video ..\..\Amgen-BW CD, 2006\PCR\Pcr.exe http://highered.mcgraw-hill.com/sites/9834092339/student_view0/chapter18/pcr_reactions.html http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf

Lab 8: PCR (Polymerase Chain Reaction)

The locus we will amplify is located in the tissue Plasminogen Activator (tPA) gene. This gene is on chromosome 8. The gene codes for a protein that is involved with dissolving blood clots. tPA is a protein given to heart attack victims to reduce the incidence of strokes.

The region we will be amplifying is located in an intron (non-translated region), of the tPA gene.

Quick Review on Exons and Introns

The intron that we will be targeting for amplification is dimorphic, which means the locus has two forms. one form carries a 300 bp DNA fragment known as an Alu element the second form of the locus does not carry this fragment.

. The diagram indicates the intron we will be targeting for PCR. Two Possibilities: 100 bp sequence 400bp sequence

What are Alu elements? Alu elements are short, around 300 bp, DNA fragments that are distributed throughout our genome. Estimated that we may carry over 1,000,000 copies of this fragment.

Karyotype with Alu tagged A probe with a florescent tag was created that was specific to the alu segment. Chromosomes were ‘denatured’ and spread on a slide, then arranged. Less than .5% are polymorphic

The PCR Reaction How does it work? Heat (94oC) to denature DNA strands Cool (56oC) to anneal primers to template Warm (72oC) to activate Taq polymerase, which extends primers and replicates DNA Repeat 40 cycles

PCR 94 C: Denature DNA 56 C: Anneal Primers to Template 72 C: Activates Taq Polymerase Repeats 31 times

The PCR Reaction What do you need? What is needed for PCR? Template - the DNA to be amplified Primers - 2 short specific pieces of DNA whose sequence flanks the target sequence Forward Reverse Nucleotides - dATP, dCTP, dGTP, dTTP Magnesium chloride - enzyme cofactor Buffer - maintains pH & contains salt Taq DNA polymerase – thermophillic enzyme from hot springs (Thermus aquaticus) The PCR Reaction What do you need?

What do we use? Reagents and supplies Genomic DNA sample (5 µL) P-20 pipette and tips Master mix I (10 µL/reaction) Thermal cycler 2.5 µL 10x PCR buffer w/o MgCl2 0.5 µL dNTP’s (10 mM) 2.5 µL Forward primer (4pM/ µL) 2.5 µL Reverse primer (4pM/ µL) 0.15 µL Taq polymerase (comes in PCR tube, must be vortexed because it is very dense) 1.85 µL ddH2O Master mix II (10 µL/reaction) 0.75 µL MgCl2 (50 mM) 9.25 µL ddH2O

Electrophoresis of PCR Sample: Make a 2% agarose gel: Calculations: 0.6 grams agarose in 30 mL 1X SB Buffer Add 3µL of orange G your PCR tube containing your amplified DNA sample Load into gel in numerical order Run gel at 135 V until the Orange G has migrated just past halfway across the gel. Stain gel with Ethidium Bromide for 10 min De-stain with water for 10 min Photograph gel Analyze results (lab 8 conclusion questions)

Expected Results of PCR Marker Homozygous Alu + Homozygous Alu – Heterozygous

Expected Results

PCR Results ‘M’ A B C D F G H I K L M ‘M’ N O P Q R T W Z ‘mystery’

Huntington Disease Trinucleotide repeats (CAGCAGCAG…) Over 40 of these repeats causes the disease PCR can be used to identify this disease

The Alu element maybe a part of the DNA coding for an RNA molecule that aids in the secretion of newly formed polypeptides from the cell. it has little if any effect on protein function unless it happens to become inserted into an exon or coding region