DNA Forensics Notes

DNA in Forensics

Introduction

• The discovery of DNA and the deciphering of its structure marked a turning point in understanding inheritance.
• In 1985, it was discovered that portions of the DNA structure are unique to each individual, similar to fingerprints.

Brief History

1953

• Watson and Crick described the double helix structure of DNA.
• In 1962, Watson and Crick received the Nobel Prize for their discovery.

1985

• Prof. Alec Jeffreys discovered variable number of tandem repeats (VNTR) in the human genome.
• VNTRs are short DNA sequences that repeat next to each other at a given locus, differing from individual to individual.
• Jeffreys developed DNA fingerprinting by analyzing restriction fragment length polymorphisms (RFLPs).

1985 (First Use)

• DNA fingerprinting was first used in an immigration case to prove a British citizen's identity.

1986

• DNA fingerprinting was first used in forensic science to verify a suspect's confession in a rape-murder case.
• The test exonerated Richard Buckland.

1987

• Robert Melias was the first person convicted on the basis of DNA evidence in the UK.

Background

• Each of the 60 trillion cells in the human body contains chromosomes.
• Arranged along the chromosomes are nearly 25,000 genes.

Deoxyribonucleic Acid (DNA)

• DNA consists of a sugar-phosphate backbone, nitrogenous bases (Adenine, Thymine, Cytosine, Guanine), and hydrogen bonds.
• Nitrogenous bases in DNA: Adenine, Guanine, Cytosine, Thymine.
• DNA is deoxyribonucleic acid; RNA is ribonucleic acid.

DNA Replication

• Leading and lagging strands are involved in DNA replication.
• Okazaki fragments are formed on the lagging strand.
• Transcription involves sense and antisense strands.
• RNA polymerase adds nucleotides to the 3' end.

DNA Transcription and Translation

• Transcription occurs in the cell nucleus.
• Translation occurs in the cytoplasm.
• mRNA carries codons for protein synthesis.
• tRNA carries amino acids.
• Ribosomes are involved in translation.

Human Genome

• The human genome is 3.2 billion nucleotides.
• Only 5% of the genome codes for proteins.
• 95% does not code for protein.
• 99.9% of DNA is identical between all humans.
• 0.1% of the genome is unique to an individual.

Applications of DNA Typing

• Personal Identification
• Forensic Caseworks
• Biological Relationship

Goals of DNA Analysis

• Create a DNA profile/DNA fingerprint that demonstrates the unique characteristics of an individual’s DNA.
  • For identification
  • For comparison

How to Accomplish DNA Analysis

1. Sample Collection
2. Extract the DNA
3. Make copies of DNA sample
4. Cut and Analyze DNA

DNA Extraction

• DNA may be extracted from biological fluids (e.g., blood, saliva), oral swabs, hairs, fluid stains, cadaveric tissues, and bones.
• DNA extraction consists of DNA purification from other cell components.
  • Red Blood Cells vs White Blood Cells

DNA Extraction Process

• Cellular lysis (by chemical and physical methods) to release DNA
• Protein removal (by protease) and lipid removal (by detergents or surfactants)
• Precipitation of DNA free from other cellular components

Common DNA Extraction Procedures

• Organic extraction: liquid-liquid extraction using phenol-chloroform and DNA precipitation by ice-cold ethanol or isopropanol.
• Spin columns purification: nucleic acid binding to a solid phase (silica) under special pH and salt conditions.
• Magnetic beads DNA isolation: polymer-coated magnetic beads bind to DNA in the presence of chaotropic salts.

Polymerase Chain Reaction (PCR)

• PCR is a sensitive and rapid procedure that permits the creation of millions of copies of DNA.
• PCR consists of repeated cycles of heating and cooling required for enzymatic replication of the DNA.
  • Denaturation: DNA strands are denatured by heat.
  • Annealing: Sample is cooled to allow primers to anneal to complementary regions.
  • Extension: Temperature is raised to enable DNA polymerase to add nucleotides (dNTPs) to extend the primer.

Tandem Repeats

• Portions of the DNA molecule contain sequences of letters that are repeated numerous times.
• More than 30 percent of the human genome is composed of repeating segments of DNA.
• These repeating sequences, or tandem repeats, act as filler or spacers between the coding regions of DNA.
• Tandem Repeats - A region of a chromosome that contains multiple copies of a core DNA sequence that are arranged in a repeating fashion.

Short Tandem Repeats (STRs)

• STRs are locations (loci) on the chromosome that contain short sequence elements that repeat themselves within the DNA molecule.
• They serve as helpful markers for identification because they are found in great abundance throughout the human genome.
• STRs normally consist of repeating sequences of three to seven bases; the entire strand of an STR is also very short, less than 450 bases long.
• STRs are much less susceptible to degradation and are often recovered from bodies or stains that have been subject to extreme decomposition.

DNA Typing Process

1. Sample
2. DNA Extraction
3. PCR
4. Separation
5. Tandem Repeat
6. Restriction Enzyme

Restriction Enzymes

• Restriction enzymes cut DNA into gene-sized pieces.

Electrophoresis

• Electrophoresis separates materials according to their migration rates on a stationary solid phase using an electrical potential.

Gel Electrophoresis

DNA Typing Using STRs

• STRs are ideal for multiplication by PCR, overcoming the limited-sample-size problem.
• Only the equivalent of 18 DNA-containing cells is needed to obtain a DNA profile.
• STR profiles have been used to identify the origin of saliva residue on envelopes, stamps, soda cans, and cigarette butts.
• When an STR is selected for analysis, the identity and number of core repeats must be defined, and the sequence of bases flanking the repeats must also be known.
• A mix of different primers aimed at different STRs will be used to simultaneously amplify a multitude of STRs (i.e., to multiplex).
• Some STR kits can simultaneously make copies of 24 different STRs.

Applications of DNA Typing (Revisited)

1. Personal Identification
2. Forensic Caseworks
   1. DNA Comparison
   2. Sex Identification
3. Biological Relationship

Sex Identification

• Females are XX, and males are XY.
• Every individual must have at least one X chromosome.
• Only males have the Y chromosomes.
• Females have twice the X chromosomes.

Applications of Y-STRs Analysis

• Sexual Assault Cases
  • Can easily determine the male component since female DNA does not contain Y-STR
  • Can determine the number of males present in a mixture.
  • Deposition of semen by an azoospermic or oligospermic males
• Criminal Paternity
• Disaster victim identification and/or missing persons
• A male individual can be identified by typing a male relative (such as a father, son, brother, uncle, etc.) who can be used as a reference.
• Ancestry

Biological Relationship

• DNA analysis can be used to establish biological relationships between individuals (e.g., mother, child, alleged fathers).

Mitochondrial DNA

• There are two types of DNA in a human cell: nuclear DNA and mitochondrial DNA.

DNA in Crime Scenes

• The human body is composed of trillions of cells, most containing a nucleus.
• A wide variety of cellular material can be recovered from crime scenes.
• Each nucleated cell contains two copies of an individual’s genome and can be used to generate a DNA profile.
• In practice, 15 or more cells are required to generate consistently good-quality DNA profiles from fresh material.

Considerations for Sample Collection

• Before collection, always take photographs
• The evidence collector must handle all body fluids and biologically stained materials with a minimum amount of personal contact.
• All body fluids must be assumed to be infectious.
• Each stained article should be packaged separately in a paper bag or a well-ventilated box.
• A portion of the unstained surface material near the recovered stain must likewise be removed or swabbed and placed in a separate package (substrate control).
• Collected swabs must not be packaged in a wet state.
• All packages containing biological evidence should be refrigerated or stored in a cool location out of direct sunlight until delivery to the laboratory.
• Blood in soil must be stored in a clean glass or plastic container and immediately frozen.

Location and Sources of DNA at Crime Scenes

Buccal Swab

• The least intrusive method for obtaining a DNA standard/reference is the buccal swab.
• Cotton swabs are placed in the subject’s mouth, and the inside of the cheek is vigorously swabbed, resulting in the transfer of buccal cells onto the swab.

DNA Laboratory Report

• Example of a DNA Laboratory Report, including case number, requesting party, suspect, victim, specimens submitted, DNA testing methods, genetic markers, results, and conclusion.

Golden State Killer Case

• In 1974, a crime spree of rape and murder began in the East Area of Sacramento, California.
• The suspect was linked to additional attacks in Contra Costa County, Stockton, and Modesto.
• Over the course of his criminal career, he is suspected of committing at least 13 murders, more than 50 rapes, and over 100 burglaries.
• Advancements in DNA testing linked several rapes and murders in 2001.
• Investigators used genetic genealogy to identify individuals based on their DNA in 2017.
• By tracing the DNA back to a family tree, investigators could narrow the pool of potential suspects considerably.
• Authorities identified 72-year-old Joseph James DeAngelo as the Golden State Killer.
• He is charged with eight counts of first-degree murder and is currently awaiting trial.