PCR - LPS.org

Some Phraseology
• Recall from general biology the heirarchy of structure of DNA:
• Humans carry 2 copies of the DNA in their cells (diploid). The
exception is sperm and eggs which contain one copy (haploid)
• The DNA is organized into chromosomes – long strands of DNA
• On the chromosomes, genes (sequences of DNA that code for a
protein) are found. The location of the gene on the chromosome is
its locus (plural: loci).
• Much of the DNA is non-coding (junk DNA) and even in protein
coding genes, there may be sequences that are cut out (introns)
before they are used to make a protein. The remaining sequences
are the exons.
• Genes are sequences of DNA – there are only 4 building blocks of
DNA (A,T,G and C), so the genes are actually sequences of these
nucleotides. The length and order of nucleotides determines the
type of protein that is produced by that gene.
• Differences exist between individuals largely in the non-coding DNA
(introns and junk DNA). DNA profiles detect and exploit these
What is PCR?
(basically a xerox machine)
• DNA replication gone crazy in a tube!
• Makes many copies of a specific target
sequence from a small amount of template
• Affects gene mapping and cloning and
DNA sequencing and detection
• Applications in the detection of specific
mutations, criminal investigations, and the
human genome
The Polymerase Chain Reaction
• The polymerase chain reaction (PCR)
produces many copies of a single gene or
piece of DNA.
• PCR requires DNA polymerase and a supply
of nucleotides for the new DNA strands.
• PCR is a chain reaction because the
targeted DNA is repeatedly replicated as long
as the process continues.
The Polymerase Chain Reaction
Amplifying the DNA of Interest
• Because most tissue samples from a crime
scene contain very little DNA, the goal is amplify,
or make many copies of the DNA of interest
• In STR analysis, you want to amplify the DNA
containing the tandem repeats and only this
• The process used is called Polymerase Chain
Reaction (PCR)
• PCR Machines, or thermocyclers, use repeated
cycles of heat and cooling to replicate the DNA
using many of the same enzymes found in cells
which facilitate DNA replication
• Ingredients:
• 1. dNTPs (nucleotides)
• 2. Buffer (to keep the pH and salt levels
• 3.Taq polymerase (heat stable DNA
• 4. Primers (short strands of DNA flanking
the gene(s) of interest – they initiate DNA
• Typical PCR reaction:
– 1 minute 95 ºC to denature DNA (does what
helicase does)
– 1.5 minutes 60-65 ºC (allows primers to
– 1 minute 72 ºC (allows Taq to add dNTPs)
• This cycle is repeated 30-40 times
produced millions of copies of the genes
or sequences of interest
Day 1
Add Master Mix
•Reaction buffer
•Electrophoresis dyes
•Taq polymerase
Day 2
Day 3
STR PCR (lots of acronyms)
• The procedure is the same for STR analysis, but recall
that each chromosome may have different numbers of
• The maternally and paternally inherited chromosomes
usually have different numbers of inserts, so the result
will be a 50/50 mix of amplified DNA with different
• For example, if you have 6 repeats from your mother and
2 from your father, you will amplify 2 different sized
pieces of DNA – one larger than the other
• In STR PCR, several different STR primers amplifying
several areas of interest simultaneously
How to amplify DNA...
How to amplify DNA...
Where does the data go?
• CODIS – Combined DNA Index System
• Uses 13 loci
• All forensic laboratories that use the CODIS system can
contribute to a national database.
• Only Mississippi doesn’t participate
• The Forensic Index contains DNA profiles from crime
scene evidence.
• The Offender Index contains DNA profiles of individuals
convicted of sex offenses (and other violent crimes) with
many states now expanding legislation to include other
– Forensic Profiles in NDIS: 119,782
– Convicted Offender Profiles in NDIS: 2,643,409
A Sample Profile
• By combining the frequency information for all
13 CODIS loci, the frequency of this profile
would be 1 in 7.7 quadrillion
Case Study: The First Use of DNA Evidence
• Two teenage girls raped and murdered in Leicestershire,
• Semen from the victims indicated a male with Type A
blood and a rare enzyme = 10% of the local male
• A local boy, Richard Buckland, confesses upon
• Police use DNA fingerprinting to confirm, but DNA
profiles of Buckland and crime scene DNA do not match
• Ironically, Buckland becomes the first person exonerated
by DNA evidence
Case Study: The First Use of DNA Evidence
• Police request DNA samples from all
adult males in 3 nearby villages (5000
• 6 months later – no results!
• A year later, police are informed by a
bakery worker that they overheard a coworker bragging they had given a DNA
sample for another man
• Police obtain DNA from Colin Pitchfork
and obtain a perfect match
The Result?
• In 1988, Colin Pitchfork was tried and
convicted and sentenced to life in prison
for the double rape and homicide based in
large part to the DNA evidence
As the technology gets smarter, so too do the criminals
• A physician in Canada eludes authorities for years
• Accused of drugging and sexually assaulting patients,
DNA profiles from semen samples from the assaulted
women do not match Dr. Schneeberger
• Blood was drawn on 3 occasions in 1992, 1993 and
1996, but never came back as a match
• Finally police obtain blood from a finger prick, swabbed
the inside of his cheek and took hair samples
• The results matched the DNA from the semen of the
• How did he get away with it?
As the technology gets smarter, so too do the criminals
• On the previous 3 occasions, blood was drawn
from the same arm
• The last time the blood was drawn, the technician
stated that the blood looked brown and “old”
• Schneeberger had surgically implanted a piece of
rubber tubing in his arm and filled it with stored
blood from a patient