Exam 2 FRSC BIO

DNA Analysis Study Guide

Historical Information

  • 1953: James Watson and Francis Crick discovered the DNA molecule's structure.

  • 1980: Ray White describes the first polymorphic RFLP marker.

  • 1985: Alec Jeffreys isolates DNA markers and calls them DNA fingerprints.

  • 1985: Kary Mullis develops PCR testing.

  • 1988: FBI starts DNA casework.

  • 1991: First STR paper published.

  • 1992: FBI begins casework with PCR (DQA1).

  • 1996: FBI starts mtDNA testing; first multiplex STR kits available.

  • 1998: FBI launches CODIS database.

  • 2001: First Y-STR kit available (analyzes specific Y chromosome locations).

  • 2003: Completion of the Human Genome Project.

  • 2016: First use of probabilistic genotyping software in court.

What is DNA?

  • Chemical blueprint of life.

  • Deoxyribonucleic Acid (DNA): hereditary material of all living organisms.

  • Found mostly in the cell nucleus.

  • Double helix structure.

DNA Structure

  • Composed of nucleotides: sugar molecule, phosphate, and nitrogen base.

  • Four nitrogen bases:

    • Cytosine (C)

    • Guanine (G)

    • Adenine (A)

    • Thymine (T)

  • Base pairing:

    • A → T

    • G → C

  • Human DNA sequence is 99.9% identical; forensic analysis focuses on the 0.1% variation.

Chromosomes and Genes

  • Most body cells have a nucleus containing chromosomes.

  • 22 pairs of autosomes + sex chromosomes (XX or XY).

  • Genes: Code for traits (eye color, hair color) and functions (immune response, cell structure).

  • Non-coding sequences (tandem repeats) are used in forensic DNA analysis.

Tandem Repeats

  • Types:

    • Satellites: Large repetitive areas, not useful in forensics.

    • Minisatellites: Hundreds of base pairs, moderate forensic use.

    • Microsatellites (STRs - Short Tandem Repeats): 2-6 base pairs, excellent forensic markers.

DNA Polymorphisms

  • Types:

    • Sequence polymorphisms (mutations in base sequences).

    • Length polymorphisms (differences in STR repeat numbers).

  • STRs are highly variable and analyzed in forensic DNA profiling.

DNA Profile Development Process

  1. Sampling

  2. Extraction

  3. Quantification

  4. Amplification (PCR)

  5. Capillary Electrophoresis

  6. Data Analysis

DNA Extraction Methods

  • Organic Extraction (Phenol-Chloroform): High purity DNA, but hazardous and time-consuming.

  • Chelex Extraction: Quick, limited application.

  • Silica-based Extraction: Automated, widely used.

  • FTA Paper: Ideal for known samples, automated.

  • Differential Extraction: Used in sexual assault cases to separate sperm and epithelial DNA.

DNA Quantification Methods

  • Yield Gel Electrophoresis: Low sensitivity, not human-specific.

  • Slot Blot: Human-specific, discontinued in 2007.

  • Real-Time PCR (qPCR): Measures DNA concentration using fluorescence.

PCR (Polymerase Chain Reaction)

  • Purpose: Amplifies small DNA segments for analysis.

  • Steps:

    1. Denaturation (94-96°C): DNA strands separate.

    2. Annealing (50-60°C): Primers bind to DNA.

    3. Extension (72°C): DNA polymerase replicates DNA.

  • Multiplex PCR: Simultaneously amplifies multiple DNA loci using different primers and fluorescent dyes.

Capillary Electrophoresis

  • Separates amplified DNA fragments by size.

  • Produces an electropherogram used in DNA profiling.

Forensic DNA Databases

  • CODIS (Combined DNA Index System): National DNA database maintained by the FBI.

  • Used to match DNA profiles in criminal investigations.