Lab 8: Lab 8: PCRPCR(Polymerase Chain Reaction)(Polymerase Chain Reaction)

By Kristi Schramm

Gabrielino HS

PCR – Polymerase Chain PCR – Polymerase Chain Reaction has many Reaction has many

applicationsapplications PCR is commonly used to PCR is commonly used to produce many produce many

copies of a selected genecopies of a selected gene segment or segment or locuslocus of of DNA.DNA.

In criminal forensics, PCR is used to amplify In criminal forensics, PCR is used to amplify DNA evidence from small samples that may DNA evidence from small samples that may have been left at a crime scene. have been left at a crime scene.

PCR can be used to amplify DNA for genetic PCR can be used to amplify DNA for genetic disease screeningdisease screening

Lab 8: Obtaining DNA Sample Lab 8: Obtaining DNA Sample

1)1) Add cheek cells to ChelexAdd cheek cells to Chelex

2)2) Boil (lyse cells and destroy nuclease)Boil (lyse cells and destroy nuclease)

3)3) Centrifuge Centrifuge

4)4) Extract DNA sampleExtract DNA sample

Lab 8: Lab 8: PCRPCR(Polymerase Chain Reaction)(Polymerase Chain Reaction)

The locus we will amplify is located in the The locus we will amplify is located in the ttissue issue PPlasminogen lasminogen AActivator (ctivator (tPAtPA)) gene. gene.

This gene is on chromosome 8. This gene is on chromosome 8.

The gene codes for a protein that is The gene codes for a protein that is involved with dissolving blood clots. involved with dissolving blood clots.

tPAtPA is a protein given to heart attack is a protein given to heart attack victims to reduce the incidence of strokes. victims to reduce the incidence of strokes.

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

Quick Review on Exons and Quick Review on Exons and IntronsIntrons

The intron that we will be The intron that we will be targeting for amplification is targeting for amplification is dimorphicdimorphic,, which means the which means the locus has locus has two formstwo forms. .

one form carries a one form carries a 300 bp300 bp DNA DNA fragment known as an fragment known as an AluAlu elementelement

the second form of the locus the second form of the locus does not carry this fragmentdoes not carry this fragment..

. . The diagram The diagram

indicates the indicates the intron we will be intron we will be targeting for targeting for PCR.PCR.

Two Possibilities: Two Possibilities: 100 bp100 bp sequence sequence 400bp400bp sequence sequence

What are Alu elements?What are Alu elements?

AluAlu elements are elements are shortshort, , around around 300 bp,300 bp, DNA DNA fragments that are distributed fragments that are distributed throughout our genome. throughout our genome.

Estimated that we may carry Estimated that we may carry over 1,000,000 copies of this over 1,000,000 copies of this fragmentfragment. .

The PCR The PCR ReactionReaction

How How does it does it work?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

1) 94 C: Denature DNA2) 56 C: Anneal Primers to Template3) 72 C: Activates Taq Polymerase

• Repeats 31 times

PCR

The The PCR PCR

ReactioReactionn

What What do you do you need?need?

What is needed for PCR?What is needed for PCR?

Template -Template - the DNA to be amplified the DNA to be amplified Primers -Primers - 2 short specific pieces of 2 short specific pieces of

DNA whose sequence flanksDNA whose sequence flanks the target the target sequence sequence

ForwardForward ReverseReverse

Nucleotides -Nucleotides - dATP, dCTP, dGTP, dTTP dATP, dCTP, dGTP, dTTP Magnesium chloride -Magnesium chloride - enzyme enzyme

cofactorcofactor Buffer -Buffer - maintains pH & contains salt maintains pH & contains salt TaqTaq DNA polymerase – DNA polymerase – thermophillic thermophillic

enzyme from hot springs enzyme from hot springs (Thermus (Thermus aquaticus)aquaticus)

What do we use?What do we use? Reagents and suppliesReagents and supplies EquipmentEquipment and suppliesand supplies Genomic DNAGenomic DNA sample (5 µL) sample (5 µL) P-20 pipette and P-20 pipette and

tipstips Master mix I (10 µL/reaction)Master mix I (10 µL/reaction) Thermal cyclerThermal cycler 2.5 µL 10x PCR buffer w/o MgCl22.5 µL 10x PCR buffer w/o MgCl2 0.5 µL dNTP’s (10 mM)0.5 µL dNTP’s (10 mM) 2.5 µL Forward primer (4pM/ µL)2.5 µL Forward primer (4pM/ µL) 2.5 µL Reverse primer (4pM/ µL)2.5 µL Reverse primer (4pM/ µL) 0.15 µL0.15 µL Taq Taq polymerase polymerase 1.85 µL ddH2O1.85 µL ddH2O Master mix II (10 µL/reaction)Master mix II (10 µL/reaction) 0.75 µL MgCl2 (50 mM)0.75 µL MgCl2 (50 mM) 9.25 µL ddH2O9.25 µL ddH2O

Expected Results of PCRExpected Results of PCR

1.1. Marker Marker

2.2. Homozygous Alu +Homozygous Alu +

3.3. Homozygous Alu –Homozygous Alu –

4.4. HeterozygousHeterozygous

Expected ResultsExpected Results

Huntington DiseaseHuntington Disease

Trinucleotide repeats Trinucleotide repeats (CAGCAGCAG…)(CAGCAGCAG…)

Over 40 of these repeats causes the Over 40 of these repeats causes the diseasedisease

PCR can be used to identify this PCR can be used to identify this diseasedisease

The The AluAlu element element maybe a part of the maybe a part of the DNA coding for an RNA molecule that DNA coding for an RNA molecule that aids in the secretion of newly formed aids in the secretion of newly formed polypeptides from the cell. polypeptides from the cell.

it has little if any effect on protein it has little if any effect on protein function unless it happens to become function unless it happens to become inserted into an exon or coding inserted into an exon or coding regionregion