BUTLER SAQ

What are the main challenges in isolating ancient DNA (aDNA)?

Degradation/Fragmentation: aDNA is short (<100 bp) and chemically damaged (e.g., cytosine deamination).

Low Abundance: Endogenous aDNA is often <5% of total DNA in a sample.

Contamination: Risk from modern human DNA or environmental microbes.

How do researchers select samples for aDNA studies?

Petrous bone (inner ear): Best preservation due to density.

Cold/dry environments: Permafrost or stable caves (e.g., Denisova Cave) reduce degradation.

What methods prevent contamination during aDNA analysis?

Clean labs: UV sterilization and positive air pressure.

Authentication:

Check for cytosine deamination (aDNA damage pattern).

Compare mtDNA/nuclear DNA ratios to detect contamination.

How is aDNA prepared for sequencing?

Library prep: Ligate adaptors to short fragments.

Enrichment: Use probes to target specific regions (e.g., mtDNA) or whole-genome sequencing.

What bioinformatics tools validate aDNA?

Mapping: Align fragments to reference genomes (human/chimp).

Damage analysis: Tools like mapDamage identify deamination patterns.

Give an example of a successful aDNA study.

The Neanderthal genome (2010) was sequenced from 40,000-year-old Croatian bones using 1.5 billion reads, despite <5% endogenous D

How does comparative genomics help identify what makes humans unique?

By comparing human/chimp genomes, scientists pinpoint:

Genetic differences (e.g., SNPs, indels, gene duplications).

Accelerated evolution in human-specific regions (e.g., brain development genes).

Regulatory changes affecting gene expression.

What is the Ka/Ks ratio, and how does it reveal selection?

Ka/Ks: Ratio of non-synonymous (amino acid-changing) to synonymous (silent) mutations.

Ka/Ks > 1: Positive selection (adaptive changes).

Ka/Ks ≈ 1: Neutral evolution.

Ka/Ks < 1: Purifying selection (conserved traits).

How does FOXP2 illustrate human-chimp differences?

FOXP2: A transcription factor linked to speech/language.

Human vs. chimp: Only 2 amino acid changes in humans (both under positive selection).

Evidence:

Mutations in FOXP2 cause speech disorders (KE family study).

Highly conserved in other primates (suggests human-specific function).

What other genomic differences distinguish humans from chimps?

Gene loss: Caspase-12 (linked to immunity/brain function).

Duplications: SRGAP2 (brain cortex development).

Regulatory changes: Hair keratin genes, lactase persistence.

Why is chromosome 2 fusion significant?

Humans have 23 chromosome pairs (vs. 24 in chimps).

Fusion of two ancestral ape chromosomes created human chromosome 2.

Evidence: Telomere remnants and centromere fossils on chr2.

What limits our understanding from comparative genomics?

Function unknown: Many human-specific changes lack clear phenotypic links.

Non-coding DNA: Regulatory regions are harder to interpret than protein-coding genes.

What do "Mitochondrial Eve" and "Y-chromosome Adam" represent?

Mitochondrial Eve: The most recent common female ancestor of all living humans (~190,000 years ago), traced through mtDNA (maternally inherited).

Y-chromosome Adam: The most recent common male ancestor (~245,000 years ago), traced through the Y chromosome (paternally inherited).

How do these concepts support the "Out of Africa" hypothesis?

Both ancestors lived in Africa, consistent with human origins there.

mtDNA/Y-chromosome diversity is highest in African populations, indicating longer evolutionary history.

Non-Africans show bottleneck effects (reduced diversity), suggesting a small founder group migrated out.

Why are mtDNA and Y chromosomes used for ancestry studies?

No recombination: Passed unchanged (except for mutations), simplifying lineage tracing.

High mutation rates: Provide clear markers for divergence times.

What genomic evidence shows interbreeding with Neanderthals/Denisovans?

Non-Africans share 1–4% Neanderthal DNA (from interbreeding ~55,000 years ago).

Melanesians have 3–6% Denisovan DNA (unique to Oceania).

Identified via SNP sharing and ancient genome comparisons.

What advantages might this DNA have provided?

Immune genes (HLA): Improved pathogen resistance (e.g., HLA-C*0702 from Neanderthals).

High-altitude adaptation: Tibetans’ EPAS1 gene from Denisovans aids oxygen use.

What are potential disadvantages?

Infertility: Some Neanderthal DNA is absent from X chromosomes (suggesting reproductive barriers).

Disease risks: Neanderthal alleles linked to severe COVID-19, depression, and autoimmune disorders.

RT-PCR Principle

Converts RNA to cDNA, then PCR-amplifies target genes.

RT-PCR Advantages

Low cost, simple.

RT-PCR Applications

Small-scale gene validation.

qPCR Principle

Quantifies cDNA in real-time using fluorescent probes (TaqMan/SYBR Green).

qPCR Advantages

High sensitivity, absolute quantification.

qPCR Applications

Clinical diagnostics (e.g., viral load).

RNA-seq Principle

Sequences all RNA fragments in a sample

RNA-seq Advantages

Genome-wide novel transcripts/splice variants.

RNA-seq Applications

Transcriptome cancer research.

When would you use RNA-seq over qPCR?

For discovery-based research (e.g., identifying new genes or isoforms), whereas qPCR is best for validating known targets.

Why is genetic diversity highest in Africa?

Older populations: Longer time for mutations to accumulate.

No bottleneck: Non-Africans descended from a smaller migrant group (~70,000 years ago).

What does linkage disequilibrium (LD) reveal?

High LD in non-Africans: Indicates recent population bottlenecks.

Low LD in Africans: Reflects large, stable populations with more recombination.

How do SNP studies support human origins?

Private SNPs: Rare in non-Africans (consistent with recent expansion).

Shared SNPs: Most alleles exist globally but at different frequencies (no "races" at the genetic level)