15 - Chromosomes and digital PCR

C value paradox

• No correlation between genome size and organism complexity

• Non coding DNA - interons interrupt genes, intergenic DNA is the spacer region between genes

Repetitive DNA

• Moderately repetitive (25%) - rRNA genes, transposons (LINES - RTase, SINES)

• Highly repetitive (10%) - satteline DNA repeats at centromeres, microsattelites used in fingerprinting

Gene families

• Clusteres - globin

• Dispersed - histone

• Pseudogenes - defective copies from mutations or retrotransposition

Chromatin organisation

• Euchromatin - transcriptionally active, less condensed

• Heterochromatin - silent, highly condensed (centromeres, telomeres), satellite DNA forms heterochromatin at centromeres

• Telomeres - TTAGGG repeats, protect chromosome ends

Mobile genetic elements

• Transposons (45%) - LTR retrosposons (viral), DNA transposons mostly inactive

• Selfish DNA - no phenotypic benefit - mostly SINEs or LINEs

• Single nucleotide polymorphism (SNP) - occur every 300bp

Digital PCR

• Sample split into thousands of wells (dPCR) or water in oil droplets (ddPCR), uses flourescence

• Lambda = mean target copy number / partition (0.6-1.6 ideal)

• Poisson statistics used to calculate the mean target copy number per partition

• Each partition contains 0, 1 or more (dilution) target DNA molecules

• Absolute quantification without standard curve

dPCR vs qPCR

• Absolute vs relative count

• Sensitive vs limited by background noise

• Tolerates inhibitors better vs affected by inhibitors

• No need for referance genes vs requires endogenous control

• Lower throughput, higher cost, requires microfluids to create high density partitions

Clinical applications

• Cancer - detects rare mutations

• Infectious diseases - quantify viral loads

• Prenatal testing - fetal DNA analysis

•Minimal residual disease - cancer reccurence