Download PCR and Forensics

PCR and Forensics
Tina Doss
Applied Biosystems
Directory for Forensic DNA Presentation
Biology Review
Nomenclature for DNA Markers
Forensic DNA – Overview
Sample Collection
PCR
References
Forensic DNA – Biology Review
DNA has two primary
purposes:
 To make copies of itself
so cells can divide and
carry on the same
information.
 To carry instructions on
how to make proteins.
Forensic DNA – Biology Review
DNA has 3 parts:
 A base
 A sugar
(pentose)
 A phosphate
group
Click picture for DNA Structure animation
Forensic DNA – Biology Review
 A DNA sequence is
normally written and
read from 5’ to 3’.
 DNA Polymerase is
the enzyme that will
“write” sequences.
 Much like we read in a
certain direction, like left
to right.
Click picture for DNA Replication animation
Forensic DNA – Biology Review
 DNA has 2 strands linked
through process called
hybridization.
 Complementary base
pairing is completed
through hydrogen bonds
between bases:
 A=T
_
 G=C
 The strands run antiparallel
Click picture for Replication Fork animation
Forensic DNA – Biology Review
 DNA may be denatured (split
2 strands) by:
 Heat DNA to near boiling
temperatures
 Place DNA in a salt solution
of low ionic strength
 Expose DNA to chemical
denaturants (ex: Urea)
Forensic DNA – Biology Review
 Denaturation is reversible.
 Process of 2 complementary DNA strands
coming back together is called
renaturation or reannealing.
 In lab, renaturation occurs during cold
cycles.
Forensic DNA – Biology Review
Approximately 3 billion
base pairs in a single
copy of human genome
Chromosome =
dense packet of DNA
wrapped around
proteins called histones
Forensic DNA – Biology Review
 Somatic (body) cells
are diploid = 2 sets of each
chromosome
 23 pairs or 46
chromosomes total
 22 pairs of autosomes
and 1 pair of sex
chromosomes
Forensic DNA – Biology Review
Genes have exons
(protein coding portions)
and introns (non
coding portions).
Markers used for human
identity testing are found
in introns either between
genes or within genes.
Forensic DNA – Biology Review
Location of a gene or
DNA marker is called a
locus.
Thousands of loci have
been characterized and
mapped to particular
regions of chromosomes
thanks to Human
Genome Project.
Forensic DNA – Biology Review
Designating Physical Chromosome Locations:
Telomere
p (short arm)
Band 2 on p-arm
Centromere
q (long arm)
Telomere
Four Squares
Write as much information
as you can about DNA
and the biology review.
_____________ is like
____________ because:
Questions or Confusions?
Applications for use or
relationship to what I know
now.
Forensic DNA – Nomenclature for DNA
Markers
If marker is part of a gene or falls
within a gene – the gene name
is used
EX: STR TH01-11
TH = human tyrosine hydroxylase
gene
01 = means the repeat region is
located within intron 1 of the
tyrosine hydroxylase gene
11 = located on chromosome 11
Forensic DNA – Nomenclature for DNA
Markers
 If marker is outside gene regions
designated by chromosomal position
the naming is slightly different:
 EX: D5S18
D = DNA
5 = Chromosome #
S = DNA marker is a single
copy in the genome
18 = indicates the historical
order in which the marker was
discovered
Forensic DNA – Nomenclature for DNA
Markers
 So what does D1S80 stand for?
 EX: D1S80
D = DNA
1 = Chromosome #
S = DNA marker is a single copy in the
genome
80 = indicates the order in which the
marker was discovered
 Where would this type of marker be found –
within the gene regions or outside the gene
regions? Outside the gene regions based on the fact
that it does not bear the name of the gene it
is located in.
Forensic DNA - Overview
Sample Collection
Purification
Quantification
STR PCR
Forensic DNA – Sample Collection
Sources of biological materials used for PCR-based DNA typing:
 Blood and Blood Stains
 Semen and Semen Stains
 Bones and Teeth
 Hair (Root and Shaft)
 Saliva
 Urine and Feces
 Debris from Fingernails
 Cigarette Butts
 Postage Stamps
 Envelope Sealing Flaps
 Dandruff
 Fingerprints
Forensic DNA – PCR
 PCR = Polymerase Chain Reaction
 Process in which a specific region of DNA is
replicated over and over again
 VNTR = Variable Number of Tandem
Repeats
 STR = Short Tandem Repeats
Forensic DNA – PCR
 The Power of PCR:
1. Only need a small
amount of DNA.
2. Lab and analysis is
completed quickly.
3. May use your own
DNA as template.
Forensic DNA – PCR
•
Need 2 primers to “flank” region of DNA
to be copied.
 Use a forward and reverse primer to start as
the starting point and isolate the target DNA
sequence.
Forensic DNA – PCR
Also need polymerase that can build
bases in the correct order from template
DNA strand.
• MgCl2 is needed for activation of the
polymerase.
•
Forensic DNA – PCR
In the Master mix:
1. Buffer – stabilizes pH for Polymerase to
work
2. MgCl2 for DNA Polymerase activation
3. dNTP (Deoxynucleoside tri-phosphate)
 These are your A, T, G and C’s
4. AmpliTaq Gold = DNA Polymerase
Forensic DNA – PCR
 Positive Control
 Valuable indicator of
whether or not any of the
PCR components failed or
were not added to the
experiment.
 Also valuable to detect if
thermal cycling
parameters are working
for amplification of DNA.
 DNA template is amplified
with the same primers.
 Negative Control
 This is the entire PCR
reaction mixture without
any DNA template. (Use
water or buffer instead of
DNA)
 Useful to assess whether
or not PCR components
are contaminated by
DNA.
Forensic DNA – PCR
•
The instrument that heats and cools a DNA sample for
PCR is called a Thermal Cycler.
Thermal Cycling Parameters:
1 HOLD
95oC
10:00
32 CYCLES
95oC
0:15
65oC
2 HOLDS
72oC
72oC
0:40
10:00
0:30
4oC
Activation
∞
During this stage, AmpliTaq Gold DNA polymerase is activated. The heat causes
the pH of the buffer to drop and the chemical modification on the polymerase to fall
off, activating the enzyme.
Forensic DNA – PCR
Thermal Cycling Parameters:
1 HOLD
95oC
10:00
32 CYCLES
95oC
0:15
65oC
2 HOLDS
72oC
72oC
0:40
10:00
0:30
4oC
Activation
Denature Stage
Heat is used instead of helicase to unwind the DNA strand.
The DNA strand is pulled apart.
∞
Forensic DNA – PCR
Thermal Cycling Parameters:
1 HOLD
95oC
10:00
32 CYCLES
95oC
0:15
65oC
2 HOLDS
72oC
72oC
0:40
10:00
0:30
4oC
Activation
∞
Denature
Annealing
This is the stage where your forward and reverse primers attach to the DNA
strand. Remember that the primers are looking for the complimentary bases
and will target on piece of DNA to amplify.
Forensic DNA – PCR
Thermal Cycling Parameters:
1 HOLD
95oC
10:00
32 CYCLES
95oC
0:15
65oC
2 HOLDS
72oC
72oC
0:40
10:00
0:30
4oC
Activation
Denature
∞
Annealing
Extension
During this stage, polymerase attaches and copies the bases from the parental DNA
strand in the 5’ to 3’ direction.
Forensic DNA – PCR
Thermal Cycling Parameters:
The denature, anneal, and extension stages repeat as many as 40 and as few
as 30 times. For D1S80, the cycle repeats 32 times.
1 HOLD
95oC
10:00
32 CYCLES
95oC
0:15
65oC
2 HOLDS
72oC
72oC
0:40
10:00
0:30
4oC
∞
Forensic DNA – PCR
Thermal Cycling Parameters:
1 HOLD
95oC
10:00
32 CYCLES
95oC
0:15
65oC
2 HOLDS
72oC
72oC
0:40
10:00
0:30
4oC
Activation
Denature
Annealing
Extension
∞
Final Extension
This stage is to allow sufficient time for all DNA fragments from previous cycles
to finish extension.
Forensic DNA – PCR
Thermal Cycling Parameters:
1 HOLD
95oC
10:00
32 CYCLES
95oC
0:15
65oC
2 HOLDS
72oC
72oC
0:40
10:00
0:30
4oC
Activation
Denature
Annealing
Extension
∞
Final Extension
Final Hold
This stage is to slow down all the processes and help keep the solution stable.
This is like putting your sample in the refrigerator.
Forensic DNA – PCR
Thermal Cycling Parameters:
 Now it’s your turn.
 Label the following stages on the cycling protocol below:
 Annealing stage, Denature stage, Extension Stage,
Final Extension Stage, Activation Stage, Final Hold
Stage.
 Make sure to add a one sentence summary for each
stage.
Forensic DNA – PCR
Thermal Cycling Parameters Answers:
Annealing Stage
Activation Stage
Denaturing
Stage
Final Extension
Extension
Stage
Final Hold
Four Squares
Write as much information
as you can about PCR
relates to DNA.
Summarize PCR:
Questions or Confusions?
Applications for use or
relationship to what I know
now.
Forensic DNA - References
 Butler, John M. (2005). Forensic DNA Typing:
Biology and Technology Behind STR Markers.
Academic Press.
 Daugherty, Ellyn (2006). Biotechnology: Science
for the New Millenium. St. Paul: Paradiigm
Publishing.
 Furtado, Manohar (2007). Lead Scientist, Foster
City, Applied Biosystems.
 Fang, Rixun (2007). Lead Scientist, Foster City,
Applied Biosystems.
 Vatta, Paolo (2007). Lead Scientist, Foster City,
Applied Biosystems.