Forensic DNA Analysis- FINALS L10

DNA

Is a molecule that is found in nearly all cells.

POLYMER

A molecule made up of repeating simpler units, called monomers

LOCATION OF DNA IN A CELL

Nucleus and mitochondria

NUCLEAR DNA

• Found in a geometric shape called a double helix.

• Made up of alternating sugar molecules (deoxyribose) and phosphates

• Dangling off each sugar molecule is one of four bases or nucleotides: adenine (A), guanine (G), cytosine (C), and thymine (T).

DNA TYPING

1. Restriction Fragment Length Polymorphism (RFLP)

2. Polymerase Chain Reaction (PCR)

RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP)

1. DNA is extracted from biologic material and then severed into small fragments called using restriction enzymes Minisatellites.

2. The length polymorphism present in these minisatellites is used to discriminate a population of people.

3. In forensic analysis, four to six of these highly polymorphic loci are analyzed

POLYMERASE CHAIN REACTION (PCR)

• Sensitive; prone to contamination

• DNA extractions are always done in a location physically isolated from the place where the subsequent amplifications will be performed

• Done in thermal cyclers

STEPS IN PCR

1. Denaturation

2. Annealing

3. Extension

DENATURATION

• The DNA added to PCR containing the reaction mixture

• Heated to 95°C. → double stranded DNA denatures.

• Bonds between the base pairs holding the strands together break, resulting in single stranded DNA

Each strand will be the template for the formation of a new piece of double-stranded DNA

ANNEALING

• Attach a short strand of synthetic DNA (called primers) to each of the separated strands.

• Primers serve as starting points for the addition of new bases to complete the reproduction of each strand.

• The thermal cycler temperature drops to 60 °C

EXTENSION

• Reaction Temperature raised to 72 °C.

• Under the influence of Taq polymerase, single bases (nucleotides) are added to the primer.

• In this way the entire complementary strand is built up,

• Double-stranded DNA is produced.

• Temperature raised to 94 °C → process repeats

DIFFERENCES BETWEEN MITOCHORNDRIL DNA AND GENOMIC DNA

• mtDNA is circular in shape

• More numerous than genomic DNA (each cell has only 2 copies)

• All male and female mtdna comes from the mother

• mtDNA often shows a high degree of variation between unrelated people, making it a powerful tool in forensic typing