PV92 PCR

PV92 PCR

Alu insert, PV92 locus, chromosome 16

What is PCR?

• DNA replication gone crazy in a tube!

• Makes many copies of a specific target sequence from a small amount of template DNA

• Affects gene mapping and cloning and DNA sequencing and detection

• Applications in the detection of specific mutations, criminal investigations, and the human genome

The target sequence…

• Your DNA!!!!• Only Chromosome 16:

Out of 23 total• Only the Alu insertion

at the PV92 locus: A 600 base pair region out of over 3 billion in your total genome!

• Key PCR Concepts: – Small amount of

template DNA– Target specific regions

of DNA– Fast results

Chromosome 16:In metaphaseChromosome 16

Human Cheek Cell

Histones

DNA Double Helix

Base Pairs

PV92 Alu insert

• A member of Alu repeat family– Repetitive sequences make up almost half of the

human genome, Alu repeats are just one kind • 306 base pair segment of DNA, Classified as a

SINE (Short Interspersed Repetitive Element)• Named for the Alu I restriction site within the

sequence• Human-specific Alu insertion• Approx. 1 million Alu copies per haploid genome

= 11% of the genome: role in genetic architecture and genetic disorders

• Intron: Found in a non-coding region of your DNA

NOT diagnostic for any disease or disorder!

Alu sequence

• GGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGCGGATCACGAGGTCAGGAGATCGAGACCATCCCGGCTAAAACGCTGAAACCTCGTCTCTACTAAAAATACAAAAAATTAGCCGGGCGTAGTGGCGGGCGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGTGAGCCGAGATCCTGCCACTGCACTCCAGCGTGGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAAAAAAA

• 306 base pairs long: This sequence remains the same, no matter where it is found in the genome

• IntronsIntrons are non- are non-

coding segments coding segments

of DNA and of DNA and

remain remain ““insideinside””

the nucleusthe nucleus

• Exons Exons are are

segments of DNA segments of DNA

that code for that code for

proteins and they proteins and they

““exitexit”” the nucleus the nucleus

Introns vs. Exons

PCR Results

– 641 bp

– 941 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 AluAlu is dimorphic so there is dimorphic so there are two possible are two possible PCR productsPCR products::

Possible Genotypes?

• Each gene locus has a particular form of the gene, or allele

• What are the possible alleles for the Alu insert at each locus?

+, Alu present

-, Alu not present

• What are the possible genotypes for the Alu insert for any given person?Homozygous positive: +/+

Homozygous negative: -/-

Heterozygous: +/-

Chromosome 16 Homologous Chromosomes

PV92 Locus

What is needed for PCR?

• Template (the DNA you want to amplify for the study)

• Sequence-specific primers flanking the target sequence

ForwardReverse

• Nucleotides (dATP, dCTP, dGTP, dTTP)

• Buffer, containing salt

• Taq polymerase

• Magnesium chloride (enzyme cofactor)

How does PCR work?

• Heat (94oC) to denature DNA strands

• Cool (60oC) to anneal primers to template

• Warm (72oC) to activate Taq polymerase, which extends primers and replicates DNA

• Repeat multiple cycles

Denaturing Template DNA

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

5’

Denaturation of DNA at 94Denaturation of DNA at 94ooCC

3’

5’

5’

3’

5’

3’

Annealing Primers

• Primers bind to the template sequence• Taq polymerase recognizes double-stranded substrate

3’

5’

5’

3’

Primers anneal at 60Primers anneal at 60ooCC3’

5’

3’ 5’

5’

3’

3’5’

Taq polymerase extends…..

• Taq polymerase extends primer• DNA 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 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/ddnalc/resources/shockwave/pcranwhole.html

PV92 locus sequence with Alu

• AACTGGGAAAATTTGAAGAGAAAGTCACACAGATACATTTCAGTAAGGTTGTCTCTGTTACTTGAGGCTTACAAGAAGGAAAGAAGGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGCGGATCACGAGGTCAGGAGATCGAGACCATCCCGGCTAAAACGCTGAAACCTCGTCTCTACTAAAAATACAAAAAATTAGCCGGGCGTAGTGGCGGGCGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGTGAGCCGAGATCCTGCCACTGCACTCCAGCGTGGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAAAAAAAGAAAGAATTCCCTCTCTAAACACACTCTAACACACAGGAGTTGAGAACTCA

• Where would your primers be if you were looking for this alu insert anywhere in the genome?

• Where would your primers be if you were looking for only the alu insert at the PV92 locus?

Actual Alu PCR Results

941 bp941 bp641 bp641 bp

-- +/-+/-++

++ -- +/-+/-