2 Historical Information w In 1953, James Watson and Francis Crick discovered the configuration of the DNA molecule w In 1980, Ray White described first.
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Historical Information In 1953, James Watson and Francis Crick
discovered the configuration of the DNA molecule In 1980, Ray White described first polymorphic
RFLP marker In 1985, Alec Jeffreys isolated DNA markers and
called them DNA fingerprints In 1985, Kary Mullis developed PCR testing In 1988, the FBI starts DNA casework In 1991, the first STR paper was written In 1998, the FBI launches CODIS database.
General DNA Information
There are approximately 60 trillion cells in the body. Each of these cells contain strands of chromosomes Chromosomes are made of genes A gene is the basic unit of heredity Each gene is composed of DNA DNA stands for deoxyribonucleic acid DNA is a polymer which is a large molecule made by
linking repeating units
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General DNA Information
The repeating units of DNA are nucleotides These nucleotides contain a sugar molecule (deoxyribose),
phosphate group and a nitrogen-containing base There are four nitrogen-containing bases:
• Adenine• Cytosine• Guanine• Thymine
The bases always pair A to T and G to C The only way that the base pairings can be properly
aligned is in a double-helix configuration
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General DNA Information
The order in which the base pairs are arranged determines the role and function of a DNA molecule
The average human chromosome has DNA that contains 100 million base pairs
DNA directs the production of proteins A protein is a polymer made of amino acids There are twenty known amino acids that are used
to make thousands of proteins
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General DNA Information
DNA replication is the process of creating a new strand of DNA from an existing strand of DNA
The process begins with the unwinding of the double helix followed by the “unzipping” of the two strands
Next, free nucleotides pair with each single strand to create two identical double helix molecule
Many enzymes and proteins are involved in this process
DNA Body Locations Can be found in all body cells--blood, semen,
saliva, urine, hair, teeth, bone, tissue Most abundant in our buccal (cheek) cells Blood is 99.9% red blood cells that have no nuclei;
and therefore, no nuclear DNA DNA obtained from blood comes from white blood
cells
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Collection and Preservation of Evidence DNA may be extracted from one cell so the
collection of even the smallest item is crucial All biological evidence should be photographed
close up and recorded in notes and sketches All body fluids and biological evidence must be
collected with a minimum of human contact Gloves should be changed frequently during the
collection process
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Collection and Preservation of Evidence All clothing from both the victim and suspect
should be collected and bagged separately Paper bags must be used for the collection of
biological evidence A substrate control must be collected. This is a
sample from an unstained portion of the object from which biological evidence has been collected
DNA evidence should be compared to the suspects DNA
Blood and buccal cell samples should be collected
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Collection and Preservation of Evidence If an individual is not available to give a
DNA sample, items should as a toothbrush, razor and comb may be collected and swabbed
Small items should be collected with clean forceps. A new pair of forceps should be used for each item
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At the Laboratory
Once the evidence has been checked into the lab, the technicians will need to extract the DNA from the objects
Contamination must be avoided so each piece of evidence will be examined separately before another piece is brought into the area
After DNA has been extracted, the process of DNA typing begins
DNA TYPINGDNA typing is a method in which DNA is
converted into a series of bands that ultimately distinguishes each individual. Only one-tenth of a single percent of DNA (about 3 million bases) differs from one person to the next. Scientists use these regions to generate a DNA profile of an individual.
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Non-Coding Regions
3 percent of the human DNA sequences code for proteins
97 percent is non-coding and is repetitive; repeating the same sequence over and over
50 percent of the human genome has interspersed repetitive sequences
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General DNA Typing Process
Obtain a DNA sample. Cut the DNA into small pieces at certain
chromosomes landmarks where there is variation from person to person.
Load and run electrophoresis. This separates the pieces by length.
Visualize the bands.
DNA TYPING“Fingerprinting”
The following are different ways to type DNA:
RFLP-- Restriction Fragment Length Polymorphism PCR-- Polymerase Chain Reaction STR--Short Tandem Repeats Mitochondrial--use of maternal DNA in the
mitochondria
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RFLP--Restriction Fragment Length Polymorphisms
Fragments are cut from the sequence of bases by a restriction enzyme.
The enzyme find its combination, bonds at one end and dissolves through the DNA at the other.
Fragments are loaded into a gel and run by electrophoresis.
DNA is extracted from the gel by blotting it into a nylon membrane.
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RFLP--Restriction Fragment Length Polymorphisms Radioactive phosphorus-32 probes are added to
the membrane which bond to the precise DNA fragments making them radioactive.
Then the membrane is placed over standard X-ray film where the radiation emitted from the P-32 gradually exposes the film and shows the DNA bands.
This process takes about 10 weeks to complete.
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PCR--Polymerase Chain Reaction
PCR is a technique for making many copies of a defined segment of a DNA molecule. It looks at six different inherited traits, each controlled by a specific gene. Every gene has at least two alternative forms called alleles. An individual receives one allele from mother and one from father. If the alleles are the same, the individual is said to be homozygous for the trait; if the two alleles are different, the individual is heterozygous.
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PCR--Polymerase Chain Reaction
Heat the DNA strands to 94 degrees which causes the strands to separate (unzip).
Add primers to the separated strands. The primers combine with the DNA strands. Add DNA polymerase and a mixture of free
nucleotides to the separated strands. The tube is cooled down to 72 degrees which
results in the rebuilding of a double-strand of DNA.
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PCR--Polymerase Chain Reaction
The outcome is a doubling of the number DNA strands. Heating, cooling, and strand rebuilding is repeated typically 25 to 30 times, yielding more than one million copies of the original DNA molecule. Each cycle takes less than two minutes from start to finish.
Advantages of PCR Minute amounts of DNA template may be used from as
little as a single cell. DNA degraded to fragments only a few hundred base pairs
in length can serve as effective templates for amplification. Large numbers of copies of specific DNA sequences can
be amplified simultaneously with multiplex PCR reactions. Commercial kits are now available for easy PCR reaction
setup and amplification.
Problem: Contaminant DNA, such as fungal and bacterial sources, will not amplify because human-specific primers are used.
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Short Tandem Repeats (STR)
STR is the latest method of DNA typing. STR’s are locations (loci) on the chromosome that contain short sequences of 3 to 7 bases that repeat themselves with the DNA molecule. This method’s advantages include a higher discrimination than RFLP, less time, smaller sample size, and less susceptible to degradation.
Short Tandem Repeats STRsCounting the number of repeats by size comparison
AGCT AGCT AGCT AGCT 4 Repeats
AGCT AGCT AGCT 3 Repeats
AGCT AGCT 2 Repeats
AGCT 1 Repeat
Largest
Smallest
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Short Tandem Repeats (STR)
Extract the gene TH01 from the sample. (TH01 has seven human variants and a repeating sequence of A-A-T-G)
Amplify the sample by means of PCR Separate by electrophoresis Examine the distance the STR migrates to
determine the number of times TH01 repeats
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Short Tandem Repeats (STR)
Each person has two STR types for TH01--one inherited from each parent.
By continuing the process with additional STRs from other genes, you can narrow down the probability of DNA belonging to only one possible person.
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STR
STR typing is visualized by peaks shown on a graph. Each represents the size of the DNA fragment.
The possible alleles are numbered for each loci.
Profiler Plus Allelic Ladders
D3S1358 FGAVWA
AMEL D8S1179 D21S11 D18S51
D5S818 D13S317D7S820
COfiler Allelic Ladders
D3S1358
AMEL
D7S820
D16S539
TH01TPOX CSF1PO
STR
Probability of Matching Previous STR
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Electrophoresis
An electrical current moves through a substance causing molecules to sort by size.
Smaller, lighter molecules will move the furthest on the gel.
Electrophoresis
Pipette the DNA.
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Electrophoresis (cont.)
Load DNA into the gel wells.
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Electrophoresis (cont.)
Run the gel.
Observe and compare bands of DNA.
Inlet (cathode)
Outlet (anode)
Capillary Electrophoresis (CE)
Argon Ion Laser
Fill with Polymer Solution
Fill with Polymer Solution
50-100 m x 27 cm50-100 m x 27 cm
5-20 kV5-20 kV
- +Burn capillary window
Data Acquisition and AnalysisData Acquisition and Analysis
DNA Separation occurs in minutes...
DNA Separation occurs in minutes...
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Three Possible Outcomes Match--The DNA profile appears the same.
Lab will determine the frequency.
Exclusion – The genotype comparison shows profile differences that can only be explained by the two samples originating from different sources.
Inconclusive – The data does not support a conclusion as to whether the profiles match.
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Types of DNA
Nuclear found in the nucleus constitutes 23 pair of
chromosomes inherited from both parents
each cell contains only one nuclei
Mitochondrial found in the cytoplasm is inherited only from
mother each cell contains
hundreds to thousands of mitochondria
can be found in skeletal remains
Nuclear DNA is present in the head of the sperm. Mitochondrial DNA is present in the tail. At conception, the head of the sperm enters the egg and unites with the nucleus. The tail falls off, losing the father’s mitochondrial DNA.
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Mitochondrial DNA Analysis of mtDNA is more:
• rigorous
• time consuming
• costly than nucleic testing of DNA
mtDNA is constructed in a circular or loop 37 genes are involved in mitochondrial energy
generation Is best used when nuclear DNA typing is not
possible
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Determining Probability
Databases are established by which one has determined how often a particular allele on a loci appears in a given population. By increasing the number of alleles on different loci the probability of having two people with the exact combination becomes astronomical.
STR Marker #Alleles Random match probability (FBI Caucasian)CSF1PO 11 0.112FGA 19 0.036TH01 7 0.081TPOX 7 0.195VWA 10 0.062D3S1358 10 0.075D5S818 10 0.158D7S820 11 0.065D8S1179 10 0.067D13S317 8 0.085D16S539 8 0.089D18S51 15 0.028D21S11 20 0.039
Product 0.000000000000001683
One in 594,059,679,247,5401 in 594 trillion
Probability of a Random Match Using 13 CODIS STR Markers
Probability Numbers:What do they mean?
1,000,000 1,000,000 millionmillion
1,000,000,000 1,000,000,000 billionbillion
1,000,000,000,000 1,000,000,000,000 trilliontrillion
1 x 101 x 101515 quadrillionquadrillion
1 x 101 x 101818 quintillionquintillion
1 x 101 x 1021 21 sextillionsextillion
1 x 101 x 102424 septillionseptillion
1 x 101 x 102727 octillionoctillion
1 x 101 x 103030 nonillionnonillion
1 x 101 x 103333 decilliondecillion
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Present Work in DNA CODIS--Computer Based DNA Information System
• A data base of DNA profiles of individuals convicted of sex crimes and other violent crime
TWGDAM--The Working Group for DNA Analytical Methods• Wrote the standards for DNA analysis that are part of a national
crime laboratory accreditation program
Innocence Project• Group of college law students work on cases where DNA may
prove innocence (or guilt)
FBI’s CODIS DNA Database
Combined DNA Index System Used for linking serial crimes and
unsolved cases with repeat offenders Launched October 1998 Links all 50 states Requires >4 RFLP markers
and/or 13 core STR markers
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The Future
Greater automation of the DNA typing process Use of SNP’s--single nucleotide polymorphism
which measures a one nucleotide change or difference from one individual to another. More sites are needed to differentiate between individuals (30 to 50 SNPs to attain the frequencies of the 13 STR loci), but it can be done with robots and automation.
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Over the years many innocent people have been wrongly convicted with no way of proving their innocence. Since the late 1980’s, DNA technology has been available. People, as a result, have been reopening cases and testing the evidence for DNA. Some have been exonerated because their DNA did not match.
DNA--The Search for InnocenceDNA implicates the guilty and exonerates the innocent--R. Saferstein
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