1. Alu insert, PV92 locus, chromosome 16
PV92 PCR
Alu insert, PV92 locus, chromosome 16

2. 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

3. The target sequence… Your DNA!!!! Only Chromosome 16: Out of 23 total
In metaphase
Human Cheek Cell
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
Histones
Base Pairs
DNA Double Helix

4. PV92 Alu insert NOT diagnostic for any disease or disorder!
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!

5. Alu sequence
GGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGCGGATCACGAGGTCAGGAGATCGAGACCATCCCGGCTAAAACGCTGAAACCTCGTCTCTACTAAAAATACAAAAAATTAGCCGGGCGTAGTGGCGGGCGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGTGAGCCGAGATCCTGCCACTGCACTCCAGCGTGGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAAAAAAA
306 base pairs long: This sequence remains the same, no matter where it is found in the genome

6. Introns vs. Exons
Introns are non-coding segments of DNA and remain “inside” the nucleus
Exons are segments of DNA that code for proteins and they “exit” the nucleus

7. The PV92 Alu is dimorphic so there are two possible PCR products:
PCR Results
The PV92 Alu is dimorphic so there are two possible PCR products:
No insertion: 641 bp
641 bp
941 bp
Alu insertion: 941 bp
641 bp
300 bp Alu insert

8. Possible Genotypes? PV92 Locus
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

9. Forward Reverse What is needed for PCR?
Template (the DNA you want to amplify for the study)
Sequence-specific primers flanking the target sequence
Forward
Reverse
Nucleotides (dATP, dCTP, dGTP, dTTP)
Buffer, containing salt
Taq polymerase
Magnesium chloride (enzyme cofactor)
Template: do not need to know sequence of desired template; only the sequence of sections on either side
Primers: need forward and reverse for double stranded DNA
Nucleotides: equal amounts of each are needed to extend the DNA
MgCl2: Enzyme cofactor needed for Taq polymerase to function
Buffer: maintains proper pH during thermal cycling
Taq polymerase: Heat stable polymerase originally found in bacteria in Yellowstone Natn’l park

10. Repeat multiple cycles
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

11. Denaturing Template DNA
Heat causes DNA strands to separate 5’ 3’ 3’ 5’
Denaturation of DNA at 94oC 5’ 3’ 3’ 5’

12. Primers anneal at 60oC Annealing Primers
Primers bind to the template sequence
Taq polymerase recognizes double-stranded substrate 5’ 3’ 3’ 5’
Primers anneal at 60oC 5’ 3’ 3’ 5’ 3’ 5’ 3’ 5’

13. Taq polymerase extends…..
Taq polymerase extends primer
DNA is replicated 5’ 3’ 5’ 3’ 3’ 5’
Extend at 72oC 3’ 5’ 3’ 5’ 5’ 3’
Repeat denaturing, annealing, and extending 40 cycles

14. The exact-length target product is made in the third cycle:3’ 5’
Cycle 1 3’ 5’ 3’ 5’
Cycle 2 3’ 5’ 3’ 5’ 3’ 5’
Cycle 3
View the PCR reaction at the Dolan DNA Learning center:

15. 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?

16. Actual Alu PCR Results
941 bp
641 bp -
+/- + + -
+/-