Download DNA Fingerprinting

DNA Fingerprinting
Sources of Biological Evidence
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Blood
Semen
Saliva
Urine
Hair
Teeth
Bone
Tissue
Characteristics of DNA
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DNA is a polymer with repeating units
called nucleotides.
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Each nucleotide has:
sugar molecule
phosphate group
nitrogen containing group (base)
Structure of DNA
DNA is a double stranded helix
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Two DNA strands
come together to
form a DNA
molecule.
Selective base pairing holds
the strands together
Adenine (A) only pairs with Thymine (T)
Cytosine (C) only pairs with Guanine (G)
Complementary Base Pairing
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Bases can occur in any sequence on one
strand. Opposite bases will be on the other
strand.
A T C C T G G C T T A T C G C
T A G G A C C GA A T A G C G
Where do proteins come from?
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Proteins are made up of units called amino acids
Order of the amino acids determines the function
and shape of the protein.
Triplet or three bases  amino acid
C-G-T
Alanine
C-T-A
Aspartate
Normal and sickle cell red blood
cells.
Genetic diseases
e.g. Sickle cell anemia: mutation in hemoglobin.
Normal sequence: C C T G A G G A G
Sickle cell anemia: C C T G T G G A G
Changes the amino acid from
glutamine  valine
Human Genome Project
Cost: $450 million (1990-2003)
 Aim: determine the order/sequence of bases
on DNA contained in all 23 chromosomes.
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Why?
DNA is “supercoiled”!
Replication of DNA
Replication of DNA
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Parent strand must unwind.
Nucleotides are assembled to make new
“daughter” strand.
Process continues until entire parent
strand is copied.
End up with two exact copies.
DNA in the Cell
chromosome
cell nucleus
Double stranded
DNA molecule
Target Region for PCR
Individual
nucleotides
Polymerase Chain Reaction
DNA (e.g. crime scene, victim, suspect)
+ DNA polymerase
+ nucleotides (A, T, C, G)
+ primers
Place in Thermal Cycler
 multiple, exact copy of original DNA
PCR Copies DNA Exponentially
through Multiple Thermal Cycles
Original DNA target region
Thermal cycle
In 32 cycles at 100% efficiency, 1.07 billion
copies of targeted DNA region are created
Polymerase Chain Reaction (PCR).
Restriction enzymes
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Restriction enzymes: “cut” DNA at specific sequences.
e.g. Eco R1, Hind III, Pst I etc.
EcoRI
SmaI
“sticky ends”
“blunt ends”
Recombinant DNA Technology
Plasmid: vehicle used to transfer the “new” DNA into any cell.
DNA Typing
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Not all of DNA sequences code for the
production of proteins
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Tandem repeats can distinguish individuals.
HLA DQ alpha system (DQA 1)
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First commercial and validated PCR-based
genetic marker system.
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DNA typing of hair, saliva, semen stains
etc.
PCR produces multiple identical
copies.
How does PCR work?
Consider,
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-G-C-T- T-C-C-A-G-C-G-A-A-G-G-T-CIdentify “primer sequences” and design primers.
Add DNA + primers + nucleotides (G,A,T,C) +
DNA polymerase.
Heat DNA (separate the strands)
Cool DNA (primers anneal and DNA
polymerase assembles new strand)
1 CYCLE = two complete identical copies of
DNA
PCR animation (http://users.ugent.be/~avierstr/principles/pcrani.html)
Short Tandem Repeats (STRs)
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locations (loci)on the chromosome that have
sequence elements that repeat themselves within
the DNA molecule.
3-7 bases in length, repeated many times.
Every person has 2 STR types for each
element…..one inherited from each parent.
Analysis requires STR’s must be identified,
number of repeats defined and sequence of bases
flanking the STR
STR Analysis and Multiplexing
1. Extract STR* (eg. THO1,ie. A-A-T-G) from
biological sample.
2. Amplify by PCR
3. Separate on electrophoresis gel
4. Examine the distance moved to estimate to
determine the number of STR repeats that exist.
*Multiplexing allows simultaneous extraction and
amplification of a number of different STRs
Multiplexing PCR
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Over 10 Markers Can Be
Copied at Once
Sensitivities to levels less than
1 ng of DNA
Ability to Handle Mixtures
and Degraded Samples
Different Fluorescent Dyes
Used to Distinguish STR
Alleles with Overlapping Size
Ranges
Federal DNA Identification Act
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1994
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Combined DNA Index System (CODIS):
facilitate the exchange of DNA typing data
among police agencies investigating violent
crimes and sexual assaults.
DNA Index System
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Mandated collection of :
 Collection of DNA samples from
convicted offenders
 Establishment of DNA databases for law
enforcement.
Mitochondrial DNA.
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Nuclear DNA (both parents) but mitochondrial
DNA (maternal lineage only).
mtDNA is circular
mtDNA only contains information for 37 genes.
2 regions (HV1 and HV2) have greatest number of
variances.
Collection and Preservation of
Biological Evidence for DNA Analysis
Photograph, notes, sketches
2. Assume all body fluids are infectious
3. Look for blood in less obvious places.
4. Packaging!
5. Refrigerate and store away from sunlight.
6. DNA control samples.
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