Download Forensic DNA Analysis

Introduction to DNA
DNA Fingerprinting and the Polymerase
Chain Reaction
The Cell
• Smallest unit of life
• Compose all living things
• The “nucleus” (one of
many organelles) contains
genetic information the
cell needs to exist and
reproduce
- most cells organize
genetic information into
chromosomes
Chromosomes
• our body’s way of
organizing all the
information that our genetic
material contains
• 23 pairs in humans
- each pair contains one from
mother and one from father
Genes
• Each chromosome
contains 100s to 1000s of
information blocks called
genes
• Each gene is the blueprint
for a specific protein in the
body
- may tell our body what color
our eyes are supposed to be,
dozens of proteins are
responsible for synthesis of ATP,
digesting food, etc, etc etc
DNA
• Each chromosome and every
gene is made of deoxyribonucleic
acid (DNA)
• DNA is a polymer of repeating
units called nucleotides
• Each nucleotide contains three
parts
- phosphate group
- sugar (deoxyribose)
- nitrogenous base
Nucleotides
Phosphate
Sugar
The nitrogenous base is always one of four molecules:
adenine
guanine
cytosine
thymine
The DNA Backbone
• Nucleotides are linked together with alternating
P-S-P-S-P……..
The DNA Double Helix
• DNA is normally
double stranded
• The two nucleotide
chains are held together
by hydrogen bonds
•A always pairs with a T
on the other strand; C
always pairs with G
The DNA Double Helix
The two strands wrap around each other to form
helical structure shown (double helix)
Functions of DNA
• Two primary functions
- transmit information from one generation to
the next
- provide blueprint for making proteins the same
way every time
Two Types of DNA
• nuclear or chromosomal DNA (inherited from mother
and father)
• mitochondrial DNA (inherited from mother only)
DNA Replication
• DNA is unwound
• An enzyme called DNA
Polymerase adds complementary
bases to “single stranded”
- A with T
- C with G
Restriction Enzymes
Enzyme that cuts DNA at specific
sequences
Recognize and binds to 6-8 nucleotide
stretch
Gel Electrophoresis
• Gel with different sized pores
agarose and acrylamide are common materials
• Load DNA samples into wells at top of gel
• Run electric current through the gel
• DNA moves due to negative charge
• Smaller bands run “faster”
Sequence Repeats in the Human Genome
• Repeat polymorphisms (satellites) are short segments of DNA that
repeat a few to thousands of times and are found at specific locations
in human DNA
• There are many types of repeat polymorphisms that occur on human
chromosomes
• Each individual will have different numbers of these repeats at each
of these spots – the numbers of repeats at each location in you are a
random combination of the repeats in your parents
• Each of these spots, or loci, are given different names. The most
common are:
variable number of tandem repeats (VNTR)
amplified fragment length polymorphism (AmpFLP)
short tandem repeats (STR)
single nucleotide polymorphism (SNP)
Sequence Repeats in the Human Genome
Variable Number of Tandem Repeats (VNTR):
repeats of 9 to 80 base pairs (bp), total length is 500 to 23,000 bp, very
specific due to length and repeats, testing is expensive and time-consuming,
degrade in older DNA samples due to random breaking of DNA strands
Amplified Fragment Length Polymorphisms (AmpFLP):
repeats of 8 to 16 bp, total length 100 to 1300 bp, shorter and less
susceptible to degradation, first loci to be used in forensic analysis
Short Tandem Repeats
repeats of 2 to 7 bases, total length 100 to 400 bp, shorter yet thereby less
susceptible to breakage, these loci are the current standard in forensic
laboratory analysis, ideal size for PCR amplification
Single Nucleotide Polymorphisms (SNP):
a single base change as a result of mutation, not commonly useful to
forensic investigators, can be potentially used to distinguish identical twins
Polymerase Chain Reaction (PCR)
• Technique devised in 1983 to amplify small amounts of DNA
• Can be performed on DNA from a single cell
- cigarette butt, a licked stamp, root of a single hair,
1/50,000 a drop of blood (0.1 microliters)
• The amplified DNA can then be used to:
- identify a suspect or victim
-determine an individual’s sex
-determine species (if not human)
PCR to Amplify a Person’s DNA
Steps Involved:
1. Isolate repeating loci from a person’s DNA using
restriction enzymes
2. Design primers – short segments of synthetic DNA
that are complementary to DNA on either side of the
VNTR regions
3. Add vast excess of the primers and heat mixture to 75 oC
This causes DNA strands to separate by breaking
hydrogen bonds between bases
4. Cool to 15 oC. Primers hydrogen bond (anneal) to
complementary strands
5. Add DNA polymerase and all four types of nucleotides.
The polymerase (enzyme used in DNA replication) will fill
in the rest of the two strands.
You now have two identical copies of the DNA you started
with.
6. Repeat steps. Heat to break hydrogen bonds. Cool to
anneal more primers (still there in vast excess). Allow DNA
polymerase to fill in the remaining strands. Two strands of
DNA become four. Etc…Etc…Etc…..
PCR
Originally, the DNA polymerase would have to be added
between each heating step because it would fall apart at 75
degrees. Now, an enzyme called Taq DNA polymerase is
added. This is a very stable enzyme isolated from bacteria
living at thermal vents in the ocean (up to 95 oC)
In just 32 rounds of PCR, 1 copy of DNA becomes 4.2 billion
copies. This would take about 3 hours to perform in lab.
PCR Animation
DNA Fingerprinting
Used to identify individuals by their repeat regions (usually
STR) regions:
Steps involved:
1. Isolate and amplify
DNA if needed
2. DNA is cleaved into
smaller pieces with
restriction enzymes
3.
DNA is separated
with gel
electrophoresis
4. DNA is transferred to a
nylon membrane
(Southern blotting)
5. A radioactive primer is
designed that will be
complementary to unique
regions (STR, etc, regions).
Add this to nylon
membrane containing DNA.
6. Wash off excess primer and
hold nylon up to a
photographic plate to
expose. The pattern will be
unique to the individual.
Clearly, suspect one is the
match…..
If all STR regions are
considered, there is a one in 3.4
billion chance of error. This
means there may be one other
person on the planet that would
be too similar to tell the
difference.
If all other satellite regions are
also considered, the chances of
error go way, way down…
1 in 53,581,500,000,000,000,000
Mitochondrial DNA
• genetic material from the mitochondria (cellular organelle
where energy is produced)
• inherited from the mother only
Advantages:
• more sensitive (less DNA needed), degrades slower than
nuclear DNA
• can be used in cases where nuclear DNA cannot (hair
without root, skeletal remains)
Disadvantages:
• all people of same maternal line will be indistinguishable
(less discriminatory)
• more work, more time consuming, more costly
CODIS – Combined DNA Index System
• National software developed by the FBI
• Distributed to local, state, and national crime labs
• All 50 states mandate inclusion of DNA fingerprint (if
available) from violent and sexually motivated crimes
• Mostly a database of STR regions
• Thousands of matches have led to the capture of criminals
that otherwise would not have been caught
This has led numerous people to suggest a national DNA
database that would include only polymorphism
information…