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

2. What can you do with the Chromosome 16 PV92 PCR kit ?
Introduce the polymerase chain reaction (PCR) technique
Apply PCR to population genetics
Directly measure human diversity at the molecular level
Compare results to published data online

3. 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

4. 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

5. 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
This is a Ya5 Alu insert: Subfamily members by diagnostic substitutions or sequence mutations accumulated over time, JSY
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!

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

7. Alu as a Mutation
Amplified in primate genomes through RNA-dependent mechanism, retroposition
Approximately one insertion every 200 new births
What does that mean????
Contribute up to 0.4% of human genetic diseases
Inserts within genes, 0.1%
Unequal homologous recombination events between Alu repeats, 0.3%
Hypercholesterolemia, α-thalassemia, BRCA1-related breast cancer

8. Duplicated retroposon
Retroposition
Retroposon mRNA
Retroposon DNA
Retroposon
RNA polymerase
Reverse transcriptase
Integration Protein
Original Retroposon
Duplicated retroposon
Transposition: Movement of gene from one chromosome to another or movement from one site to another; does not require homology
Transposons: mobile genetic elements that enable genes to move between non-similar sites
Retroposition: Creates genetic diversity
Retroposons: Replicate and move to other sites on DNA through an RNA intermediate

9. Homologous Recombination
Parent DNA duplexes align at similar sequences and new DNA is formed by breaking and joining of homologous sequences.
RecA protein (ATP-powered) mediated through catalyzing strand exchange
Key role in DNA repair

10. 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

11. Evolutionary Significance of Alu
Some inserts are recent enough that they remain polymorphic
Highly conserved
Inserted in the last 1,000,000 years
Used in population genetics, paternity analysis, and forensics
Ancestral state known to be absent of Alu insertion
Alu insert reflects a single, unique event in human evolution

12. 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

13. 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

14. DNA Replication in Eukaryotes

15. 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

16. 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

17. View the molecular structure of DNA (see the 5’ to 3’ structure):

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

19. 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’

20. 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

21. 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:

22. 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?

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

24. PCR Procedures
Day 1
Day 2
Day 3
Day 4

25. Genomic DNA Extraction
InstaGene = Chelex® cation exchange resin; binds cellular MgCl2
56oC loosens connective tissue and inactivates DNases
100oC ruptures cell membranes and denatures proteins
InstaGene Matrix: DNases, which break down DNA, need cations like Mg++ to function. The beads of chelex bind these cations which prevents DNases from breaking down the DNA we wish to extract

26. InstaGene matrix binds released cellular Mg++
InstaGene Extraction
Cell membrane
Nuclear membrane
Mg++
Genomic
DNA
Mg++
Mg++
Heat disrupts membranes
Mg++
Mg++
InstaGene matrix binds
released cellular Mg++
Mg++

27. Extensions Making DNA probes Gene expression studies
Detection of viral pathogens or bacterial infections
Genetic diagnoses
Crime scene investigations
Anthropology
Key words: minimal template and FAST

28. Microbial Diagnostics
RFLP (a): Banding patterns are compared to reference strains
PCR (b): sensitivity allows small sample size and early detection; clinical microbiology diagnostics and microbe infections
DNA sequencing (c): Microbes affecting public health (76 million cases of food-borne disease causing over 300,00 hospitalizations per year in the US

29. DNA Fingerprinting
MLP’s (20-30 bands) ensure identification: 1 in 30 billion chance of a match between individuals
In the United States, the FBI incorporates 13 sites on average into its profiles. With 26 different bands studied, you'd be incredibly hard pressed to find two unrelated individual with the same DNA profile; the odds of a match in this case are well more than one in a hundred billion. The bottom line is that, unless you have a twin, you're statistically two thousand times more likely to win the Publisher's Clearinghouse sweepstakes (1 in 50,000,000) than to have a DNA profile that matches anyone else.