DNA
Introduction
DNA: Stands for deoxyribonucleic acid.
Discovery:
First discovered in 1868, but its role in inheritance wasn't understood until later.
In the 1950s, James Watson and Francis Crick discovered the structure of DNA, marking a major advancement in genetics.
Function of DNA: Controls genetic traits of all living cells.
DNA Fingerprinting
Alec Jeffreys:
First used DNA fingerprinting in 1985.
Investigated human genes, leading to the discovery that portions of DNA structure are unique to certain genes.
Applications: Developed techniques for DNA profiling and DNA typing.
Structure of Genetic Material
Location:
Found in the nucleus of every cell in the body.
Arranged in chromosomes.
Composition:
DNA is the genetic material that carries instructions for specific bodily functions.
Serves as the fundamental unit of heredity.
Role: Instructs the body to produce proteins.
Structure of DNA
Nucleotides:
DNA made of repeating units called nucleotides.
Each nucleotide consists of:
Sugar: Contains an OH group at the 3’ end.
Phosphorous-containing group: Located at the 5’ end.
Nitrogen-containing base: Four types are associated with DNA: adenine (A), thymine (T), guanine (G), cytosine (C).
DNA Double Helix
Structure:
DNA consists of two strands twisted together in a double-helix formation.
Strands demonstrate complementary-base pairing: A pairs with T and G pairs with C (A-T and G-C).
Complementary Strands Example
Example:
If one strand has the sequence TATTGTAAGTCA, the complementary strand would be:
A-T, T-A, T-T, G-C, T-A, A-T, G-C, T-C, A-T.
Thus, complementary strand is: A T A A C T A T T C A G T.
DNA Sequencing
Definition:
Procedure for determining the order of base pairs in DNA.
Importance:
The order of bases defines the role and function of a DNA segment.
Average human chromosome has approximately 100 million base pairs; human DNA is composed of millions of base pairs.
Function of DNA
Role in Protein Production:
DNA is responsible for synthesizing proteins composed of 20 different amino acids.
The order of amino acids determines the protein's shape and functionality.
Minor changes in amino acid sequences can lead to significant functional changes in proteins.
Example of impact: A single amino acid substitution in hemoglobin can convert normal hemoglobin to sickle cell hemoglobin.
Central Dogma of Molecular Biology
Processes:
DNA can replicate itself.
DNA is transcribed into RNA (RNA is single-stranded).
RNA is translated into proteins (using ribosomes).
Transcription
Process:
Converts double-stranded DNA to single-stranded RNA.
Involves DNA polymerase which separates DNA strands and reads one strand to synthesize RNA from the 5’ to the 3’ end.
Translation
Procedure:
RNA strand transcribed from DNA is read.
Each group of three nucleotides (codon) codes for a specific amino acid.
Amino acids are added sequentially until a stop codon is reached.
Impact of Nucleotide Changes
Example:
If a nucleotide changes, for example, substituting T for A in G-A-G to G-U-G, the protein will misincorporate amino acids thus altering its functionality, potentially leading to diseases and health issues.
DNA Replication
Occurrence:
Happens in regular cellular life to provide new cells with DNA.
Steps:
Unwind the DNA to use each strand as a template.
Each new DNA molecule consists of one parental strand and one newly synthesized strand.
Enzymes in Replication
Key Enzymes and Functions:
Various enzymes are required to:
Unwind DNA.
Maintain separation of DNA strands.
Assemble new DNA strands correctly.
DNA Polymerases:
Enzymes that synthesize new DNA strands based on the parent strand in the 5’ to 3’ direction.
Proofread the new strands to correct mismatched base pairs.
Polymerase Chain Reaction (PCR)
Purpose:
Technological method to replicate DNA ex vivo, focusing on specific DNA segments (primers).
Allows for the creation of thousands of copies from a minimal DNA sample (as little as 1 ng will yield a good profile).
Basic Measurements:
1 cell contains approximately 7 pg of DNA; need around 156 cells for a reliable profile (as few as 18-20 cells can result in acceptable profiles).
Process:
Exponential increase with each cycle,
Each template produces a new copy of DNA.
Phases of PCR
Three Phases Involved:
Each phase is conducted at a specific temperature:
Denaturation at 94°C: DNA strands separate into single strands.
Annealing at 60°C: Short synthetic DNA primer attaches to each separated strand, marking replication starting points.
Extension at 72°C: Taq polymerase adds nucleotides to the primer to form a new piece of double-stranded DNA.
Repetition in PCR
Repetition of Cycles: The three-step cycle is repeated until sufficient DNA is produced.
28-32 cycles take around 3 hours to yield 1 billion DNA copies.
Tandem Repeats in Forensic Analysis
Definition: Portions of DNA organized into repetitive units, which may provide insight into ancestry or personal identity despite not coding for any proteins.
Prevalence: Accounts for about 30% of the human genome, but their functions remain largely unknown.
Relevance in Analysis: Essential for forensic DNA profiling as they are inherited and exhibit high variability among individuals, reducing legal and ethical issues associated with gene coding information.
Types of Tandem Repeats
VNTR (Variable Number of Tandem Repeats):
15-30 bp per repeating unit and can repeat extensively (up to 1000 times).
STR (Short Tandem Repeats):
3-7 bp per unit and typically repeat 5-30 times.
Restriction Fragment Length Polymorphism (RFLP) Analysis
Process:
First method utilizing VNTRs for comparison.
Involves the use of restriction enzymes which cut DNA at specific sequences (think of them as DNA scissors).
Separation Method:
Fragments are separated by gel electrophoresis, taking advantage of DNA’s negative charge to sort based on size and charge.
Gel Electrophoresis
Method Overview:
Electric potential is applied to separate DNA fragments of varying sizes on a gel-coated plate.
Longer fragments migrate more slowly than shorter fragments, allowing for visual identification of DNA characteristics.
Transition from RFLP to STR Analysis
RFLP Challenges:
Initial scientific acceptance as a forensic protocol but became less useful due to difficulties with long DNA strands and PCR technology’s limitations.
Current Practices: Focus has shifted to Short Tandem Repeats (STRs) as they are more suitable for PCR amplification.
Capillary Electrophoresis for STR Analysis
Method Difference:
In capillary electrophoresis, separation occurs through a narrow capillary tube as opposed to a gel, still using the principles of charge and size for differentiation.
Electropherogram: Visualization of the results showing individual peaks for STR repeats.
DNA Profile Construction
STR Profiles: Full DNA profiles consist of data from multiple STR locations, typically 15 STRs for analysis. Peaks indicate the number of repeats at each locus.
Importance of Multiple Loci: The more loci analyzed, the more distinct the profile, facilitating better discrimination among individuals.
Statistical Analysis of STRs
Significance: Essential to determine how likely it is that two random individuals share the same profile characteristics.
Calculative Approach: Product rule employed, multiplying the frequencies of STR types to ascertain the probability of encountering a specific combination in a population.
Example Calculation: For three loci with frequencies of 0.055, 0.009, and 0.088, the combined frequency of occurrence is:
Profile Frequency Examples: As STRs are added, the rarity of the profile increases, example statistics reflect:
Using 6 loci: 1 in 2 million.
Using 9 loci: 1 in 69 billion.
Using all 13 STRs: 1 in 1.6E21 trillion for Caucasian Americans.
Biological Sex Determination in Forensic DNA
Using Amelogenin Gene:
Comprised of two versions on X and Y chromosomes, allowing differentiation based on band production during electrophoresis (XX for females, XY for males).
Y-STRs Usage: Analyzing Y chromosome-specific markers useful in male-only related cases.
Mixtures in DNA Sampling
Challenge: Detection of multiple contributors possible in profiles with varying peak counts.
Mitochondrial DNA (mtDNA) Overview
Types of DNA: Human cells contain nuclear DNA and mtDNA (inherited solely from mothers).
Function of Mitochondria: Powerhouses for energy production in the body (approximately 90% of energy needs).
mtDNA Structure: Circular configuration, distinct from linear nuclear DNA, includes hypervariable regions for sequencing analysis.
Sensitivity of mtDNA: Crucial for analysis when nuclear DNA is scarce or degraded.
DNA Databases and CODIS
Purpose: Comparison-based matching systems for crime scene profiles against known sources.
CODIS System:
Developed by the FBI for maintaining local, state, and national DNA databases.
As of now includes over 470,000 profiles.
Case Study Example: 1990s serial crimes in NC were linked to a suspect through reanalysis of historical DNA evidence via CODIS.
DNA Evidence Collection Practices
Best Practices:
Collect samples not directly but through potential carriers that could contain DNA (blood, skin, etc.).
Use sterile gloves and change them frequently to mitigate contamination risks.
Packaging and Handling of Biological Evidence
Guidelines:
Avoid plastics/airtight containers due to moisture accumulation risks.
Use breathable paper packaging and attach biohazard labels for identified biological evidence.
Store biological samples in a cool place to mitigate microbial degradation.
Contamination Prevention Techniques
Protective Equipment: Use of disposable gloves, lab coats, and masks recommended to minimize contamination risks.
Precautions: Change gloves between evidence handling procedures and employ disposable tools like forceps for sampling.