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Historical Information James Watson and Francis Crick--1953 discovered

the configuration of the DNA molecule Ray White--1980 describes first polymorphic

RFLP marker Alec Jeffreys--1985 isolated DNA markers and

called them DNA fingerprints Kary Mullis--1985 developed PCR testing 1988--FBI starts DNA casework 1991--first STR paper 1998--FBI launches CODIS database.

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

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. Amplify DNA sample if needed 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”

**Amplify DNA by PCR First RFLP-- Restriction Fragment Length

Polymorphism STR--Short Tandem Repeats Mitochondrial--use of maternal DNA in the

mitochondria

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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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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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

My STR’s

Probability of Matching My STR’s

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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 Fingerprinting

Forensics Missing Persons Paternity/Maternity Genetics

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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.

Problems?

Ethics involved in DNA Fingerprinting for health• Insurance companies• Job Screening• Lethal Diseases

Legal issues arising from genome sequencing• Use of “I’m genetically predisposed”

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WORDS OF WISDOM

Keep in mind a famous quote from Albert Einstein, “ If we knew what we were doing, it would not be called research, would it?”