PV92 PCR
Alu insert, PV92 locus,
chromosome 16
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
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
Human Cheek Cell
Chromosome 16
Chromosome 16:
In metaphase
Histones
Base Pairs
DNA Double Helix
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
– 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!
Alu sequence
• GGCCGGGCGCGGTGGCTCACGC
CTGTAATCCCAGCACTTTGGGAGG
CCGAGGCGGGCGGATCACGAGGT
CAGGAGATCGAGACCATCCCGGC
TAAAACGCTGAAACCTCGTCTCTA
CTAAAAATACAAAAAATTAGCCGGG
CGTAGTGGCGGGCGCCTGTAGTC
CCAGCTACTTGGGAGGCTGAGGC
AGGAGAATGGCGTGAACCCGGGA
GGCGGAGCTTGCAGTGAGCCGAG
ATCCTGCCACTGCACTCCAGCGTG
GGCGACAGAGCGAGACTCCGTCT
CAAAAAAAAAAAAAAAAAAAAAAAA
A
• 306 base pairs long: This sequence
remains the same, no matter where it
is found in the genome
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
Retroposition
• 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
Retroposon
RNA polymerase
Retroposon mRNA
Reverse
transcriptase
Retroposon DNA
Integration Protein
Original
Retroposon
Duplicated
retroposon
Homologous Recombination
• Parent DNA duplexes align
at similar sequences and
new DNA is formed by
breaking and joining of
homologous sequences.
• RecA protein (ATPpowered) mediated
through catalyzing strand
exchange
• Key role in DNA repair
Introns vs. Exons
• Introns are noncoding segments
of DNA and
remain “inside”
the nucleus
• Exons are
segments of DNA
that code for
proteins and they
“exit” the nucleus
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
PCR Results
• The PV92 Alu is dimorphic so there
are two possible PCR products:
– 641 bp
No insertion: 641 bp
Alu insertion: 941 bp
641 bp
– 941 bp
300 bp Alu insert
Possible Genotypes?
• Each gene locus has a
Chromosome 16
particular form of the gene, or
Homologous Chromosomes
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?
PV92 Homozygous positive: +/+
Homozygous negative: -/Locus Heterozygous: +/-
DNA Replication in Eukaryotes
http://www.johnkyrk.com/DNAreplication.html
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)
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
View the molecular structure of DNA (see the 5’ to 3’ structure):
http://207.207.4.198/pub/flash/24/menu.swf
Denaturing Template DNA
Heat causes DNA strands to separate
5’
3’
3’
5’
Denaturation of DNA at 94oC
5’
3’
3’
5’
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’
Taq polymerase extends…..
• Taq polymerase extends primer
• DNA is replicated
5’
3’
5’
3’
3’
5’
Extend at 72oC
3’
5’
3’
3’
5’
5’
Repeat denaturing, annealing, and extending 40 cycles
The exact-length target product is made in the third cycle:
5’
Cycle 1
Cycle 2
Cycle 3
3’
5’
3’
5’
3’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
5’
5’
3’
3’
5’
5’
3’
View the PCR reaction at the Dolan DNA Learning
center:
http://www.dnalc.org/ddnalc/resources/shockwave/pcranwh
ole.html
PV92 locus sequence with Alu
• AACTGGGAAAATTTGAAGAGAAAGTCACACAGATACAT
TTCAGTAAGGTTGTCTCTGTTACTTGAGGCTTACAAGAA
GGAAAGAAGGCCGGGCGCGGTGGCTCACGCCTGTAAT
CCCAGCACTTTGGGAGGCCGAGGCGGGCGGATCACG
AGGTCAGGAGATCGAGACCATCCCGGCTAAAACGCTG
AAACCTCGTCTCTACTAAAAATACAAAAAATTAGCCGGG
CGTAGTGGCGGGCGCCTGTAGTCCCAGCTACTTGGGA
GGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCG
GAGCTTGCAGTGAGCCGAGATCCTGCCACTGCACTCC
AGCGTGGGCGACAGAGCGAGACTCCGTCTCAAAAAAA
AAAAAAAAAAAAAAAAAAGAAAGAATTCCCTCTCTAAAC
ACACTCTAACACACAGGAGTTGAGAACTCA
• 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 bp
641 bp
+ -
+/-
PCR Procedures
Day 1
Day 2
Day 3
Day 4
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 Extraction
Cell membrane
Nuclear membrane
Mg++
Genomic
DNA
Mg++
Mg++
Heat disrupts membranes
Mg++
Mg++
Mg++
InstaGene matrix binds
released cellular Mg++
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
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 foodborne disease causing
over 300,00
hospitalizations per
year in the US
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.
• http://www.dnai.org/d/index.html