Download Chapter 13 Forensic DNA

Chapter 13
Forensic DNA
Students will understand DNA’s
role in Forensics science
Students will know the different
procedures for DNA profiling
I. DNA: A Forensic Science Tool
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A. General Concepts
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1. DNA is Deoxyribonucleic Acid
2. The human genome project determined
portions of the DNA structure of certain genes
may be as unique as fingerprints.
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Discovered by Alec Jeffreys et. al. at Leicester
University, England
Called it DNA fingerprinting
 Also called DNA profiling and DNA typing
I. DNA: A Forensic Science Tool
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B. What is DNA
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1.
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3.
4.
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Chromosomes
Almost 100,000 genes per chromosome
Genes are the fundamental unit of heredity
DNA construction
Polymer, a large molecule made by linking together a
series of repeating units.
Nucleotides are the linked molecules with one
phosphate, one sugar, and one nitrogen base.
It has a double helix shape with A bonded to T and C
bonded to G
I. DNA: A Forensic Science Tool
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C. DNA at work
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1. DNA is identical due to Replication
2. Inherited traits are viewed by the
production of specific proteins
3. Proteins are made up of a combination of 20
amino acids
4. Amino Acid sequences aid in determining
the genetic code
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Codons (3 base pairs) match with tRNA anticodons
which are connected to a single amino acid
This codon can be traced back to the DNA codes for
the entire protein
I. DNA: A Forensic Science Tool
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5. Single base changes can change the entire
protein ex. Hemoglobin and sickle cell
(glutamate to valine)
I. DNA: A Forensic Science Tool
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D. DNA Replication
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1. DNA unwinds
2. New nucleotides attach to the “free”
nucleotides
3. Once the entire strand is unwound and all of
the “free” nucleotides are matched, there are
two exact DNA copies
4. Scientists can copy DNA outside of the cell
using DNA Polymerases
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Called PCR (Polymerase Chain Reaction)
Accomplished in a DNA Thermal Cycler
each cycle doubles the DNA
After 30 cycles there can be over a billion strands
DNA Polymerase: Unzipping DNA
DNA Polymerase: Unzipping DNA
I. DNA: A Forensic Science Tool
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E. Recombinant DNA
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1. Cutting and splicing sections of DNA
2. This can take advantage of known sections of DNA
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3. Recombinant DNA relics on certain chemicals to cut
DNA (called restriction enzymes)
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Know the specific site of production
The fragments can be incorporated into another DNA
strand
There are 150 commercially used restriction enzymes
4. Insert the DNA fragment into a bacterium strand with
a different restriction enzyme (called splicing)
5. Alters the bacteria’s DNA
Recombinant DNA
Recombinant DNA
Electro-Gel
Electrophoresis
Electrophoresis
I. DNA: A Forensic Science Tool
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F. DNA Typing
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1. Restriction Fragment Length Polymorphism
(RFLP)
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a. Not all DNA segments code for proteins
b. Some segments occur as repeated letter
sequences
 Called “Tandem repeats”
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c. Forensics use tandem repeats to identify subjects
 Called DNA Typing
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d. The number of repeating sequences within each
pair may be different
 Demonstrating differences between the strands from
both parents
I. DNA: A Forensic Science Tool
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F. DNA Typing (cont)
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d. (cont)
 Also in a population the number of repeating strands are
numerous
 Increases with greater number of chromosomes
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e. Restriction enzymes can be used to cut chromosomes
into different fragments with different lengths
 Are called restriction fragment length polymorphisms (RFLPs)
 Long in length
 Fragments are sorted by electrophoresis
 Small fragments move faster than large fragments
 The double stranded fragments are treated for separation
 Fragments are transferred to Nylon membrane called
Southern Blotting
RFLP process from start to finish
RFLP
Restriction Fragment Length
Polymorphism (RFLP)
RFLP: Determine the relationships
I. DNA: A Forensic Science Tool
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F. DNA Typing (cont)
 e. (cont)
 Labeled with radioactive base pairs to identify
complimentary strands (hybridization)
 X-rayed to find pairs
 When comparing look for matching bands
 First scientifically accepted protocol in the U.S.
I. DNA: A Forensic Science Tool
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F. DNA Typing (cont)
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2. Polymerase Chain Reaction (PCR)
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A second technique
More viable
Increased sensitivity
 As little as 1 billionth of a gram of DNA
 50 times less
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DNA polymerase can synthesize a specific region of
DNA
PCR
I. DNA: A Forensic Science Tool
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F. DNA Typing (cont)
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2. Polymerase Chain Reaction (PCR)
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A DNA primer is used to prep the segment
 Heat DNA strand (94°C)
 DNA separates
 Add specific sequences (primer) to the separate
strands
 Combine to the strands, while lowering the temp.
 Add DNA polymerase and some free nucleotides
 Reheat the tube (72°C)
 Polymerase directs the rebuilding of double
stranded DNA
 Extends the primers
I. DNA: A Forensic Science Tool
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F. DNA Typing (cont)
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2. Polymerase Chain Reaction (PCR) [cont]
 Doubles the number of DNA strands
 Continue doubling process until significant number of
copies are present
 Makes genetic typing easier
 First use was with the HLA DQ alpha system, (now
DQA1)
 Has significant numbers of variants
 The process is easier than RFLP
 DNA is extracted
 Primer, DNA polymerase and free nucleotides are
added
PCR
PCR
I. DNA: A Forensic Science Tool
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F. DNA Typing (cont)
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2. Polymerase Chain Reaction (PCR) [cont]
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Heated and cooled
Select DNA is added to Nylon strips
“probes” are added (look for DQA1 variants)
DQA1 genes will hybridize with probes
Looks like a blue dot for each DQA1 gene
DQA1 frequencies are greater in a population
DQA1 or HLA DQ alpha system
I. DNA: A Forensic Science Tool
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F. DNA Typing (cont)
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2. Polymerase Chain Reaction (PCR) [cont]
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Polymarker (PM)
 Type of PCR
 Types 5 additional genetic markers
 Used in combination with DQA1
 Can work saliva on cigarette butts
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3. Short Tandom Repeats (STR)
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a. General information
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Most recent method
Most successful and widely used
High discrimination and reduced period of time
STR’s are locations (loci)
 Short sequences that repeat themselves
 Found in great abundance
STR
STR
Short Tandom Repeats (STR)
I. DNA: A Forensic Science Tool
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F. DNA Typing (cont)
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3. Short Tandom Repeats (STR)
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a. General information
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Usually 3 to 7 bases
Entire strand is less than 400 bases (very short)
Less susceptible to degradation
Easier to collect, especially exposed samples
 ex) THO1 is A-A-T-G
 (tab) amplified by PCR
 (tab) separate with electrophoretic gel
 (tab) count the number of THO1
I. DNA: A Forensic Science Tool
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F. DNA Typing (cont)
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3. Short Tandom Repeats (STR)
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a. General information (cont)
 THO1 (cont)
 Hundreds of STR’s
 The more identified, the smaller the percent of
people with the same combination
 (indent) multiplexing- using more than 1 STR
during PCR process
 (indent)(indent) Ex. STR Blue Kit, uses 3 STR’s
The 13 CODIS STRs and Probability of
Identities
STR
African
American
U.S. Caucasian
D3S1358
0.097
0.080
vWA
0.074
0.068
FGA
0.036
0.041
TH01
0.114
0.080
TPOX
0.091
0.207
CSF1PO
0.079
0.128
D5S818
0.121
0.166
D13S317
0.139
0.081
D7S820
0.087
0.067
D8S1179
0.080
0.069
D21S11
0.042
0.041
D18S51
0.032
0.032
D16S539
0.076
0.091
STR
THO1
I. DNA: A Forensic Science Tool
Developed capillary electrophoresis for STR
identification
 (indent) carried out in a glass column
 Sexing comes from the amelogenin gene on the x
and y
 (indent) shorter on the x than y (6 bases)
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4. Mitochondrial DNA
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a. General concepts
 Second type of DNA
 Written as mtDNA
 Inherited solely from the mother
 Found in all cells (hundreds to thousands per cell)
I. DNA: A Forensic Science Tool
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4. Mitochondrial DNA
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a. General concepts (cont.)
 Convert sugar into ATP
 Contains several loops of DNA per mitochondria
 Can get DNA from relative, when no DNA exists from a
person long since dead
 More rigorous, time consuming, and costly
 Few labs run this type of testing
 Very few crimes are “allowed” to use this type (costbenefit ratios)
 mtDNA is circular (like bacterial DNA)
 Two regions are “highly” variable
I. DNA: A Forensic Science Tool
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4. Mitochondrial DNA
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a. General concepts (cont.)
Called Hypervariable region 1 (HV1) and
Hypervariable region 2 (HV2)
 Several labs have created an mtDNA database
 (indent) sample size isn’t large enough to have
complete statistical data
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5. Combined DNA Index System
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a. General concepts
 Will be used to look at sex crimes against criminal
databases
 All 50 states have legislation to collect DNA convicted
from sex-offenders
I. DNA: A Forensic Science Tool
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b. Combined DNA Index System
 Called CODIS
 Standardized 13 STR’s
 Created from the 50 states’ databases
Gel Electrophoresis
II. Collection and Preservation of
Biological Evidence
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A. General Concepts
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1. Evidence must be collected in the
appropriate manor
2. Position and shape of biological material can
be crucial pieces of evidence
3. Should always assume the biological
evidence is infectious
4. Should attempt to find towels or rags, in
case of hand wiping
5. Should not put into airtight containers
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Bacteria can grow and destroy evidence
Fungi as wells
II. Collection and Preservation of
Biological Evidence
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6. Placed in a paper bag and refrigerated
7. Blood (dried) should be moved with a cotton
swab dipped in distilled water (dH2O)
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Nearby areas are swabbed as a control
8. Use luminol to observe unseen blood stains
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Does not inhibit STR’s