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 from template DNAMakes many copies of target sequence 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 sequenceSequence-specific primers flanking the target sequence  Forward  Reverse Nucleotides (dATP, dCTP, dGTP, dTTP)Nucleotides (dATP, dCTP, dGTP, dTTP) Magnesium chloride (enzyme cofactor)Magnesium chloride (enzyme cofactor) Buffer, containing saltBuffer, containing salt Taq polymerase (heat stable)Taq polymerase (heat stable)

How does PCR work? Heat ( 94 o C ) to denature DNA strandsHeat ( 94 o C ) to denature DNA strands Cool ( 60 o C ) to anneal primers to templateCool ( 60 o C ) to anneal primers to template Warm ( 72 o C ) to activate Taq polymerase, which extends primers and replicates DNAWarm ( 72 o C ) to activate Taq polymerase, which extends primers and replicates DNA Repeat multiple cyclesRepeat multiple cycles

Denaturing Template DNA Heat causes DNA strands to separate 3’ 5’ 3’ Denaturation of DNA at 94 o C 5’ 3’ 5’

Annealing Primers Primers bind to the template sequencePrimers bind to the template sequence Taq polymerase recognizes double-stranded substrateTaq polymerase recognizes double-stranded substrate 3’ 5’ 3’ Primers anneal at 60 o C 3’ 5’ 3’ 5’ 3’ 5’

Taq polymerase extends….. 3’ 5’ 3’ 5’ 3’ 5’ Extend at 72 o C 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Taq polymerase extends primerTaq polymerase extends primer DNA is replicatedDNA is replicated Repeat denaturing, annealing, and extending 40 cycles

The exact-length target product is made in the third cycle 3’ 5’ 3’3’ 3’3’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Cycle 1 Cycle 2 Cycle 3 3’ 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 (Short INterspersed Repetitive Element)Classified as SINEs (Short INterspersed Repetitive Element) About 300 base pairs long repeated Approx. 500,000 times throughout the genome, representing about 5% of the genomeAbout 300 base pairs long repeated Approx. 500,000 times throughout the genome, representing about 5% of the genome Named for the Alu I restriction site within the elementNamed for the Alu I restriction site within the element Function unknownFunction unknown Useful as a measure of genetic variation, associated with disease or used for DNA typingUseful as a measure of genetic variation, associated with disease or used for DNA typing

The target sequence PV92 Alu insertion PV92 Alu insertion Found on chromosome 16 Found on chromosome 16 5’ 3’ Alu Amplified Region

PV92 Alu insertion A member of Alu repeat familyA member of Alu repeat family Human-specific Alu insertionHuman-specific Alu insertion Found in a non-coding region of your DNAFound in a non-coding region of your DNA NOT diagnostic for any disease or disorderNOT diagnostic for any disease or disorder 5’ 3’ Alu Amplified 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, paternity analysis, and forensicsUsed in population genetics, paternity analysis, and forensics

PCR Results The PV92 Alu is dimorphic so there are two possible PCR products:The PV92 Alu is dimorphic so there are two possible PCR products: 641 bp 941 bp No insertion: 641 bp 300 bp Alu insert 641 bp Alu insertion: 941 bp 5’ 3’ Alu Amplified Region

Actual Alu PCR Results 941 bp 641 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 56 o C Incubate at 100 o C

Genomic DNA extraction Genomic DNA extraction ¶ InstaGene = Chelex ® cation exchange resin; binds cellular MgCl 2 binds cellular MgCl 2 Inactivates DNases as Mg 2+ are cofactors · 56 o C loosens connective tissue and inactivates DNases ¸ 100 o C ruptures cell membranes and denatures proteins releasing DNA

InstaGene Extraction Cell membrane Nuclear membrane Heat disrupts membranes InstaGene matrix binds released cellular Mg ++ Genomic DNA DNA 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 56 o C 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 –94 o C 1 min - denature –60 o C 1 min – anneal primers –72 o C 1 min – extend primers –40 cycles After PCR run samples on agarose gel