Forensic DNA Analysis

DNA

Nucleotides:

Phosphate

Sugar

Base

Restriction endonucleases

Also called restriction enzymes

Occur naturally in bacteria

Hundreds are purified and available commercially

Named for bacterial genus, species, strain, and type

Recognize specific base sequences in DNA

Cut DNA at those recognition sites

Restriction Enzyme Recognition Site

Enzymes recognize specific 4-8 bp sequences

Recognition sites have symmetry

Some enzymes cut in a staggered fashion

Types of unknown samples

Blood, Semen, Stains, Saliva

Hair, Tissue, Bones, Teeth

Types of known samples

Blood or buccal swabs from suspect or victim or other known person

Contamination

Contamination occurs when DNA from another source gets mixed in with the sample being collected.

An investigator touches, sneezes, bleeds on a sample.

Wear gloves and use disposable instruments

Package items separately.

Especially, do not mix known samples (from victim or suspect) with unknown samples.

Packaging Evidence

Package each item individually.

Put evidence into paper bags, not plastic.

Moisture degrades DNA; air dry samples.

Keep samples at room temperature and out of sun.

Technologies used to analyze DNA

RFLP

PCR

STR

Y-STR

SNP

Mitochondrial DNA Analysis

DNA fingerprint

Every cell of an individual carries a copy of the DNA

a cell collected from a person's skin or hair folicle contains the same DNA as from that persons heart tissue or white blood cells

Order of base pairs in the DNA of every individual is different except identical twins

Determination of an individual's unique

collection of DNA restriction fragments

Restriction Fragment Length Polymorphism

Analyzes variable lengths of DNA fragments

One of the original applications of DNA analysis

requires a large quantity of DNA sample

samples degraded by the environment do not work well

testing requires a relatively large amount of HMW DNA (~50ng = thousands of cells)

Not ideal for forensic evidence, in which small, degraded samples are common

Polymerase Chain Reaction

molecular Xeroxing

Three temperature phases, carried out in a Thermal Cycler, replicate or "amplify" the desired DNA fragment(s)

Preparation of a DNA fingerprint

Specimen collection

blood, semen, etc

easy to contaminate a DNA sample with DNA from other sources (bacteria, DNA of person collecting sample)

DNA is not stable for very long-it degrades

sunlight

heat

moisture

Basic Steps in Analysis

Extraction

Amplification or PCR

Separation

Sequencing

Extraction

Separates DNA from sample

need to separate DNA from other cell material and debris from crime scene.

Amplification or PCR

Amplifies small portions of DNA (STR regions)

Separation

Separates amplified fragments according to size.

PCR amplification of DNA

1 strand of DNA

Heat to denature double-stranded DNA

Design primers that anneal to STR locus

Amplify all the regions of the chromosome

where the STRs exist.

allows you to make millions of copies of the STR region from a single copy of DNA you recovered from crime scene.

FMBIO

Separates and Measures Amplified DNA

Gel Electrophoresis

Black and white image of STR gel.

Samples will have one or two bands at each loci.

Sequencing

Sequence of letters for amplified fragments

Short Tandem Repeats

-Currently the most used of all forensic markers

-Individual identification possible

-Type of data used in the FBI CODIS database

-People differ in length at these loci

-Are located in the nuclear DNA (chromosomes)

-Number of repeats varies greatly between individuals

-make up 10-15% of the mammalian genome

-are also called "microsatellites"

-are "junk DNA

Each person has some ___that were inherited from mother and some from father

No person has ____ that are identical to those of either parent

The number of repeats at each loci on chromosome is highly variable in the population, ranging from 4 to 40.

The length of the DNA after cutting the chromosome with a restriction enzyme, and its position after electrophoresis will depend on the exact number of repeats at the locus

Simple Tandem Repeats

STR - region of DNA containing tandem copies

of di-, tri- or tetranucleotide repeat units.

Locus/Loci

Refers to the location on the chromosome.

Allele

Refers to the type of DNA.

For STRs, will be the number of repeats

Combined DNA Index System

largest DNA database

is divided into two indices: the forensic index and the offender index

Dot Blot

Genes that display high sequence variability can be substituted for STRs

An example of a gene with this property is the human leukocyte antigen (HLA).

Much sequence variability across the human population.

not present in other life forms, it reduces the interference that could otherwise be contributed by bacteria, fungi, dog, or cat DNA picked up in the sample at crime scene.

A visual signal is produced when the different probes anneal (bind) to the complementary sequence in the DNA sample

show a pattern of spots that either light up or remain dark

Compare pattern produced from crime scene DNA to pattern produced from suspect DNA

Southern Blotting

-method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples

-Other blotting methods (i.e., western blot,[2] northern blot, eastern blot, southwestern blot) that employ similar principles, but using RNA or protein

Y-STRs

Test for markers found only on the Y-chromosome. Only male DNA is amplified!

Single Nucleotide Polymorphisms

Point mutations (base substitutions) found in 1% or more of the population