PV92 PCR/Informatics KitPV92 PCR/Informatics Kit

Alu insert, PV92 locus, Alu insert, PV92 locus, chromosome 16chromosome 16

What can you do with the What can you do with the Chromosome 16 PV92 PCR kit ?Chromosome 16 PV92 PCR kit ?Introduce the polymerase chain reaction (PCR) Introduce the polymerase chain reaction (PCR) techniquetechnique

Apply PCR to population geneticsApply PCR to population genetics

Directly measure human diversity at the Directly measure human diversity at the molecular levelmolecular level

Compare results to published data onlineCompare results to published data online

What is PCR?What is PCR?

DNA replication gone crazy in a tube!DNA replication gone crazy in a tube!Makes many copies of a specific target Makes many copies of a specific target sequence from a small amount of template sequence from a small amount of template DNADNAAffects gene mapping and cloning and Affects gene mapping and cloning and DNA sequencing and detectionDNA sequencing and detectionApplications in the detection of specific Applications in the detection of specific mutations, criminal investigations, and the mutations, criminal investigations, and the human genomehuman genome

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

TemplateTemplate (the DNA you want to amplify for the (the DNA you want to amplify for the study)study)

Sequence-specific Sequence-specific primersprimers flanking the target flanking the target sequence sequence

ForwardForwardReverseReverse

NucleotidesNucleotides (dATP, dCTP, dGTP, dTTP) (dATP, dCTP, dGTP, dTTP)

Magnesium chlorideMagnesium chloride (enzyme cofactor) (enzyme cofactor)

BufferBuffer, containing salt, containing salt

Taq polymeraseTaq polymerase

How does PCR work?How does PCR work?

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

Cool (Cool (6060ooCC) to anneal primers to ) to anneal primers to templatetemplate

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

RepeatRepeat multiple cycles multiple cycles

Denaturing Template DNADenaturing Template DNA

Heat causes DNA strands to separateHeat causes DNA strands to separate3’

5’

Denaturation of DNA at 94Denaturation of DNA at 94ooCC

Annealing PrimersAnnealing Primers

Primers bind to the template sequencePrimers bind to the template sequenceTaq Taq polymerase recognizes double-stranded polymerase recognizes double-stranded substratesubstrate

3’

5’

5’

3’

Primers anneal at 60Primers anneal at 60ooCC3’

5’

3’ 5’

5’

3’

3’5’

TaqTaq polymerase extends….. polymerase extends…..

TaqTaq polymerase extends primer polymerase extends primer

DNA is replicatedDNA is replicated

Extend at 72Extend at 72ooCC

3’

5’3’ 5’3’5’

5’

3’

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

3’ 5’3’5’

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

3’

5’3’5’3’5’

5’

3’Cycle 1

3’

5’5’

3’

5’

3’

5’

3’Cycle 2

3’

3’

3’

3’5’

5’

5’

5’

Cycle 3

View the PCR reaction at the Dolan DNA Learning View the PCR reaction at the Dolan DNA Learning center:center:

http://www.dnalc.org/shockwave/pcranwhole.html

The target sequenceThe target sequence

PV92 PV92 AluAlu insertion insertion

Found on chromosome 16Found on chromosome 16

5’5’ 3’3’Alu

Amplified RegionAmplified Region

PV92 PV92 AluAlu insertion insertion

A member of A member of AluAlu repeat family repeat family

Human-specific Human-specific AluAlu insertion insertion

Intron: Found in a non-coding region of your DNAIntron: Found 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

picture, Copyright © 2002 Pearson Education, Ipicture, Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummingnc., publishing as Benjamin Cumming

Introns vs. ExonsIntrons vs. Exons

IntronsIntrons are non- are non-

coding segments coding segments

of DNA and remain of DNA and remain

“inside”“inside” the the

nucleusnucleus

ExonsExons are are

segments of DNA segments of DNA

that code for that code for

proteins and they proteins and they

“exit”“exit” the nucleus the nucleus

AluAlu repeatsrepeats

Classified as SINEs (Classified as SINEs (SShort hort InInterspersed terspersed Repetitive Repetitive EElement)lement)

Approx. 500,000 Approx. 500,000 AluAlu copies per haploid copies per haploid genome, representing about 5% of the genome, representing about 5% of the genomegenome

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

Evolutionary Significance of PV92 Evolutionary Significance of PV92 AluAlu InsertsInserts

Highly conservedHighly conserved

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

Used in population genetics, paternity Used in population genetics, paternity analysis, and forensicsanalysis, and forensics

PCR ResultsPCR Results

641 bp641 bp

941 bp941 bp

No insertion: 641 bpNo insertion: 641 bp

300 bp 300 bp Alu Alu insertinsert

641 bp641 bp Alu insertion: 941 bpAlu insertion: 941 bp

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

Actual Actual Alu Alu PCR ResultsPCR Results

941 bp941 bp641 bp641 bp

-- +/-+/-++

++ -- +/-+/-

PCR Procedures Day 1Day 1

Day 3Day 3

Day 2Day 2

Add Master Mix containing:

•Nucleotides•Primers•Reaction buffer•Electrophoresis dyes•Taq polymerase

Genomic DNA ExtractionGenomic DNA Extraction

InstaGeneInstaGene = Chelex= Chelex®® cation exchange cation exchange resin;binds cellular MgClresin;binds cellular MgCl22

5656ooCC loosens connective tissue and loosens connective tissue and inactivates DNasesinactivates DNases

100100ooCC ruptures cell membranes and ruptures cell membranes and denatures proteinsdenatures proteins

InstaGene ExtractionInstaGene ExtractionCell 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++

ExtensionsExtensions

Population GeneticsPopulation Genetics

InformaticsInformatics