Chromosome 16: PV92 PCR. What is PCR? DNA replication gone crazy in a tube!DNA replication gone crazy in a tube! Makes many copies of target sequence.

Chromosome 16:Chromosome 16:PV92 PCRPV92 PCR

What is PCR?

• DNA replication gone crazy in a DNA replication gone crazy in a tube!tube!

• Makes many copies of target Makes many copies of target sequence from template DNAsequence from template DNA

What is needed for PCR?• Template (the DNA you want to amplify for the study)Template (the DNA you want to amplify for the study)• Sequence-specific primers flanking the target Sequence-specific primers flanking the target

sequence sequence ForwardForwardReverseReverse

• Nucleotides (dATP, dCTP, dGTP, dTTP)Nucleotides (dATP, dCTP, dGTP, dTTP)

• Magnesium chloride (enzyme cofactor)Magnesium chloride (enzyme cofactor)

• Buffer, containing saltBuffer, containing salt• TaqTaq polymerase (heat stable) polymerase (heat stable)

How does PCR work?

• Heat (Heat (9494ooCC) to denature DNA strands) to denature DNA strands

• Cool (Cool (6060ooCC) to anneal primers to template) to anneal primers to template

• Warm (Warm (7272ooCC) to activate ) to activate TaqTaq polymerase, polymerase, which extends primers and replicates DNAwhich extends primers and replicates DNA

• RepeatRepeat multiple cycles multiple cycles

Denaturing Template DNA

Heat causes DNA strands to separateHeat causes DNA strands to separate

3’

5’

5’

3’

Denaturation of DNA at 94Denaturation of DNA at 94ooCC

5’

3’

3’

5’

Annealing Primers•Primers bind to the template sequencePrimers bind to the template sequence

•Taq Taq polymerase recognizes double-stranded substratepolymerase recognizes double-stranded substrate

3’

5’

5’

3’

Primers anneal at 60Primers anneal at 60ooCC3’

5’

5’

3’3’ 5’3’5’

Taq polymerase extends…..

3’

5’3’ 5’3’5’

Extend at 72Extend at 72ooCC

3’

5’3’ 5’3’5’

5’

3’

5’

3’

•TaqTaq polymerase extends primer polymerase extends primer

•DNA is replicatedDNA is replicated

RepeatRepeat denaturing, annealing, and extending denaturing, annealing, and extending 40 cycles40 cycles

The exact-length target product is made in the third cycle

3’

5’3’5’3’5’

5’

3’

3’

5’5’

3’

5’

3’

5’3’

Cycle 1

Cycle 2

Cycle 33’

3’

3’

3’5’

5’

5’

5’

What are we going to amplify?

The genome contains small repetitive DNA elements that have become randomly inserted into the human genome over millions of years

Alu repeats

• Classified as SINEs (Classified as SINEs (SShort hort ININterspersed Repetitive terspersed Repetitive EElement)lement)

• About 300 base pairs long repeated Approx. 500,000 times About 300 base pairs long repeated Approx. 500,000 times throughout the genome, representing about 5% of the genomethroughout the genome, representing about 5% of the genome

• Named for the Named for the AluAlu I restriction site within the element I restriction site within the element

• Function unknownFunction unknown

• Useful as a measure of genetic variation, associated with Useful as a measure of genetic variation, associated with disease or used for DNA typingdisease or used for DNA typing

The target sequence

• PV92 PV92 AluAlu insertion insertion

• Found on chromosome 16Found on chromosome 16

5’5’ 3’3’Alu

Amplified RegionAmplified Region

PV92 Alu insertion

• A member of A member of AluAlu repeat family repeat family

• Human-specific Human-specific AluAlu insertion insertion

• Found in a non-coding region of your DNAFound in a non-coding region of your DNA

• NOTNOT diagnostic for any disease or disorder diagnostic for any disease or disorder

5’5’ 3’3’Alu

Amplified RegionAmplified Region

Evolutionary Significance of PV92 Alu Inserts

• Highly conservedHighly conserved

• Inserted in the last 1,000,000 yearsInserted in the last 1,000,000 years

• Genotypes (+/+, +/Genotypes (+/+, +/--, , --//--))

• Used in population genetics, Used in population genetics, paternity analysis, and forensicspaternity analysis, and forensics

PCR ResultsPCR Results

• The PV92The PV92 Alu Alu is dimorphic is dimorphic so there are two possible so there are two possible PCR products:PCR products:

641 bp641 bp

941 bp941 bp

No insertion: 641 bpNo insertion: 641 bp

300 bp 300 bp Alu Alu insertinsert

641 bp 641 bp Alu insertion: 941 bp Alu insertion: 941 bp

5’5’ 3’3’Alu

Amplified RegionAmplified Region

Actual Actual Alu Alu PCR ResultsPCR Results

941 bp941 bp641 bp641 bp

-- +/-+/-++

++ -- +/-+/-

Protocol

• Preparation of template DNA• Amplification of specific sequence• Gel Analysis• Calculating gene frequency within class• Bioinformatics – comparing class frequency

to other populations

Preparing Template DNA

• Source – your cheek cells– Scrape cheeks to obtain cells– Isolate DNA

Isolating DNA

• Suspend cheek cells in instagene matrix• Incubate at 56oC• Incubate at 100oC

Genomic DNA extractionGenomic DNA extraction InstaGene = ChelexInstaGene = Chelex®® cation exchange resin; cation exchange resin;

binds cellular MgClbinds cellular MgCl2 2

Inactivates DNases asInactivates DNases as MgMg2+2+ are cofactors are cofactors

5656ooC loosens connective tissue and inactivates C loosens connective tissue and inactivates DNasesDNases

100100ooC ruptures cell membranes and denatures C ruptures cell membranes and denatures proteins releasing DNAproteins releasing DNA

InstaGene ExtractionInstaGene Extraction

Cell membraneCell membrane

Nuclear membraneNuclear membrane

Heat disrupts membranesHeat disrupts membranes

InstaGene matrix binds InstaGene matrix binds released cellular Mgreleased cellular Mg++++

GenomicGenomic DNADNA

Mg++

Mg++

Mg++

Mg++

Mg++

Mg++

Take Care and note the changes

• Instagene must be well mixed prior to use• Use a blue tip with the end cut off• Longer incubation at 56oC• Vortex mixture

PCR

• Intact genomic DNA is used as a template for PCR – ensure you do not transfer any instagene to the PCR reaction

• Master mix contains all the other components including tracking dye

Controls (per class)

• +/+ control DNA• -/- control DNA• +/- control DNA

• No DNA

Positive Controls

Negative control (contamination)

Contamination causes problems with PCR, ideally different labs should be used for preparing and analysing samples

PCR

• PCR– 94oC 1 min - denature– 60oC 1 min – anneal primers– 72oC 1 min – extend primers– 40 cycles

After PCR run samples on agarose gel