DNA testing creates DNA testing creates patternspatterns unique to unique to each individual.each individual.

Key Idea:

1.1. What is DNA?What is DNA?

2.2. How does DNA make me unique?How does DNA make me unique?

3.3. How is DNA used in profiling?How is DNA used in profiling?

4.4. Why is “junk” DNA used in forensic Why is “junk” DNA used in forensic profiling?profiling?

Goals:

Historical Information 1953--Watson and Crick discovered the structure of

the DNA molecule 1980--Ray White described the 1st polymorphic RFLP

marker 1985--Alec Jeffreys isolated DNA markers and called

them DNA “fingerprints 1985--Kary Mullis developed PCR testing 1988--FBI starts DNA casework 1991--First STR paper 1998--FBI launches CODIS database 2003--Completion of the Human Genome Project

DNA Structure Double helix--two coiled DNA strands Nucleotides--each unit contains a sugar

(deoxyribose), a phosphate group and a nitrogen-containing base

Four Bases• Adenine

• Cytosine

• Guanine

• Thymine

Bases always pair A--T and C--G

DNA Locations in the body 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

Non-Coding Regions 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.

97% is non-coding and is repetitive

3% of the human DNA sequences code for proteins

50% of the human genome has these interspersed repetitive sequences

DNA TYPING METHODS

DNA typing methods create patterns that ultimately distinguish each

individual.

KEY TO UNDERSTANDING

• The key to understanding DNA typing lies in the knowledge that within the world’s population numerous possibilities exist for the number of times a particular sequence of base letters can repeat itself on a DNA strand.

Saferstein, Richard. Criminalistics An Introduction to Forensic Science

Forensic Testing will take advantage of the repeating sections of DNA

1. Looks at the tandem repeats

2. There are many versions of some of these repeats (polymorphic)

3. Remember: Half of the DNA is from the mother, half from the father

DNA TYPING“Fingerprinting”

Methods RFLP-- Restriction Fragment Length

Polymorphism PCR-- Polymerase Chain Reaction

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

mitochondria• y-STR-only found in males

Restriction Fragment Length Polymorphism

Using restriction enzymes, differing lengths of chromosome fragments are separated by electrophoresis.

A DNA fragment pattern will be created by electrophoresis

RFLP-Restriction Fragment

Length Polymorphisms

1. Extract the DNA--from biological sample (blood, semen, saliva)

2. Add restriction enzymes--which will cut the DNA at specific regions

3. Use electrophoresis--to separate the DNA fragments by size

4. Detect regions--using luminescent dyes or radioactive probes that will expose X-ray film

RFLP-Restriction Fragment Length Polymorphisms

Takes at least 10 weeks Is very specific if multiple restriction

enzymes are used Requires a substantial sample size The large number of base units

(1000’s) per fragment make the fragments susceptible to breaking

PCR--Polymerase Chain Reaction

PCR is a technique for making many copies of a defined segment of a DNA molecule. It looks at several regions of repeated bases.

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.

PCR--Polymerase Chain Reaction steps

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.

PCRThe outcome is a doubling of the number DNA

strands. Repeat 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.

Short Tandem Repeats (STR) STR typing uses the PCR method

STR’s are locations (loci) on the chromosome that contain short sequences of 3 to 7 bases that repeat themselves with the DNA molecule.

Advantages include a higher discrimination than RFLP, less time, smaller sample size, and less susceptible to degradation.

In Forensic Science, 13 locations are used.

CODIS: Combined DNA Index System

Example: Using 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

Example con’t Each person has two STR types for TH01--

one inherited from each parent.

Continue the process with additional STRs from other genes

Imagine how the probability of a match DECREASES by using an INCREASING number of STRs.

STR Analysis

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

DNA Interactive

Go to the website below to see a STR animation demonstration. Click on human identification , profiling and then on the third circle called Today’s DNA Profiling

http://www.dnai.org/d/index.html

Electrophoresis

An electrical current moves through a substance causing molecules to sort by size.

Smaller, lighter molecules will move the furthest on the gel.

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.

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...

Gel ElectrophoresisGel Electrophoresis Capillary ElectrophoresisCapillary Electrophoresis

capillarieslaser

gel plate

DNA Analyst

at work

DNA Analyst

at work

Computer Screen

Data from capillary electrophoresis

Computer Screen

Data from capillary electrophoresis

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A comparison of the computer screen and a possible print out that represents the fragments by peaks.

Electrophoresis

Pipette the DNA.

We are working with a smaller budget and less expensive equipment. This is what we will do in class.

Electrophoresis (cont.)

Load DNA into the gel wells.

Electrophoresis (cont.)

Run the gel.

Observe and compare bands of DNA.

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Types of DNANuclear DNA

Nuclear found in the nucleus

Constitutes 23 pair of chromsomes inherited from both parents

Each cell contains only one nuclei

Mitochondrial DNA

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.

Mitochondrial DNA

37 genes are involved in mitochondrial energy generation

Is best used when nuclear DNA typing is not possible

Analysis of mtDNA is more: Rigorous Time consuming Costly than nucleic testing of DNA

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Comparison of DNA Methods

Determining Probability

Databases are established by determining 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.

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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

Present Work in DNA

CODIS--Combined 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 DatabaseCombined 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

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.

DNA--The Search for Innocence• DNA implicates the guilty and exonerates the innocent--R.

Saferstein

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.

In this unit you will research a case in which DNA was used to convict or vindicate the suspect. You will work with a partner and will present your findings to the class using PowerPoint.

• http://www.dnai.org/d/index.html

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?”