DNA Sequencing and Mutations

• DNA sequencing determines the order of nucleotide bases.

Sequencing Types

• Pyrosequencing detects light as nucleotides are added.
• Bisulfite converts unmethylated cytosines to uracil.
• Next-generation sequencing (NGS) allows for high-speed, parallel genomic analysis.

NGS Techniques

• Targeted panel sequencing focuses on disease-related genes.
• Whole-exome sequencing identifies rare genetic disorders.

NGS Library Preparation

• Involves DNA fragmentation.
• Adaptor ligation attaches short adaptors to fragment ends.
• PCR is used for library amplification.
• Size selection chooses desired fragment lengths.

DNA Mutations

• Are permanent changes to DNA.
• Genome mutations change chromosome number.
• Chromosomal mutations alter chromosome structure.
• Gene mutations affect a single gene's sequence.

Chromosome Structure

• Telomeres are protective chromosome ends.
• Centromeres are spindle fiber attachment sites.
• Nucleosomes consist of DNA wrapped around histones, forming chromatin.

Karyotyping

• Displays chromosomes by size and structure to detect abnormalities.
• Sample preparation uses phytohemagglutinin to stimulate cell division.
• Colcemid stops cells in metaphase for clear visualization.

Staining Methods

• Giemsa-banding creates unique chromosome banding patterns.
• Reverse-banding highlights GC-rich regions.
• Centromere-banding stains centromeres.
• The amelogenin gene, located on both X/Y chromosomes, determines gender based on length differences.

Fluorescence In Situ Hybridization (FISH)

• Uses fluorescent probes to identify DNA,
• Metaphase FISH detects large structural abnormalities
• Chromosome painting uses multiple probes to visualize entire chromosomes
• Interphase FISH examines non-dividing cells.
• Interphase FISH detects aneuploidy and gene rearrangements for prenatal screening.

Types of PCR

• Commercially available PCR is standard for common pathogens.
• Home-brew PCR is used for rare pathogens without FDA-approved tests.
• Multiplex PCR amplifies multiple sequences using multiple primers.
• Random amplified polymorphic DNA (RAPD) uses short primers to amplify random DNA fragments.

PCR Internal Controls

• Homologous extrinsic controls check amplification efficiency by adding a known template.
• Heterologous extrinsic controls ensure proper DNA extraction by adding a non-target DNA sequence.
• Heterologous intrinsic controls confirm specimen collection by amplifying a human gene.

Other DNA Techniques

• LAMP (Loop-Mediated Isothermal Amplification) allows for fast DNA amplification at a single temperature for point-of-care testing.
• Syndromic testing is a single test to detect multiple pathogens associated with a specific syndrome.
• SSP-PCR (Sequence-Specific Primer) detects mutations using primer extension, where a 3' primer must match the target.
• Mitochondrial DNA (mtDNA) is maternally inherited, using hypervariable regions for forensic analysis.

Molecular Test Targets

• Molecular testing may be suited when there is difficult isolation (ex. Mycobacteria), or hazardous organisms (ex. Histoplasma, Coccidioides).
• Molecular testing is used in instances of unreliable testing (ex. HIV, HCV), or in high-volume testing (ex. Streptococcus pyogenes, Neisseria gonorrhoeae, Chlamydia trachomatis).

Bacterial Strain Typing

• Pulsed-Field Gel Electrophoresis (PFGE) compares DNA bands for outbreak tracking, resolving large fragments.
• Related samples on a PFGE typically have ≤6 band differences due to a Rule of Three.
• Arbitrarily Primed PCR (RAPD) generates random DNA patterns for comparison.

DNA Polymorphism

• Is a sequence variation found in 1-2% of the population.
• Is used in forensic and paternity testing, especially with STRs
  • Southern blot measures fragmenz size
  • PCR amplifies, capillary electrophoresis reads amplicon size
  • Multiplex PCR works on multiple loci

RFLP (Restriction Fragment Length Polymorphism)

• Identifies DNA sequence variations by analyzing fragment size differences created by restriction enzymes, which is then detected by Southern blotting.

SNP (Single Nucleotide Polymorphism)

• Is a single base change (~every 1,250 bases), 99% of which have no effect.
• May be detected by sequencing, or melt curve analysis

Tandem Repeats

• Tandem repeats are regions of DNA where nucleotide sequences are repeated in a row.
• VNTR (Variable Number Tandem Repeats) have longer repeat sequences (8-50) and are used for forensic and paternity testing.

STR (Short Tandem Repeats)

• STRs are highly variable between individuals, making them ideal for identity testing.
• Y-STRs are inherited as a haplotype (no recombination) from father to son.
• Mini-STRs recover information from degraded DNA samples.

Bone Marrow Engraftment Testing

• Bone marrow engraftment testing uses STRs to track donor vs. recipient DNA ratios.
• %Recipient = (AR / (AR+AD)) x 100, where AR is the recipient peak and AD is the donor peak.