01 - basics & techniques Flashcards
Name 3 genes involved in epigenetic/chromatin & disorders associated with them
1) Cohesion - Cornelia de lange
2) MLL (KMT2A) - Acute Leukaemia
3) MECP2 - Rett Syndrome
Name 2 disorders associated with telomere malfunction
1) Dyskaratosis Congenita
2) Cri-du-Chat (5p del) - involves the hTERT gene
Describe Bloom Syndrome. How do we test for it?
> Rare AR disorder
Primordial dwarfism, sensitive skin, characteristic facies
BLM gene
Mutations in BLM result in hyper-recombination between sister chromatids = increased SCE
Test: replication staining. Treat cells initially with thymidine and later switch to Brdu (thymidine analogue). Process for chromosome staining. Chromosomes which replicate early in S phase incorporate thymidine stain dark, whereas those replicating late, incorporate Brdu and appear light.
SCE visible as harlequin chromosomes. Count number of SCE
) x10 MORE SCE in Bloom syndrome
What genes are involved in G1 checkpoint, what is their role?
> Cyclins, CDKs, RB1, E2F
Cell cycle growth enables CDK-Cyclin D formation
leads to hyperphosphorylation of Rb1
Rb1 normal inhibition of E2F is removed
E2F is a transcription factor which is now able to promote expression of Cyclin E
Cyclin E - CDK2 formation can occur
Transition into S phase
Name 3 disorders due to defects in non-coding RNA
1) Schwachmann Diamond Syndrome (SDS) - affects many parts of body, notably Bone Marrow = reduction in white blood cell production (neutropenia). At risk of developing MDS (myelodysplastic syndrome)
2) MELAS - mutations in the mt-tRNA Leu (MTTL1) gene. mostly m.3243A>G
3) MERRF - mutations in the mt-tRNA Lys gene. mostly m.8344A>G
name 3 mechanisms of mutation generation & provide an example of each
1) DNA damage:
UV radiation can cause linked pyrimidines (C/T) - thymidine dimers
2) Defects in DNA repair:
Mismatch repair pathway (MMR)
Complexes Mutsa (MSH2/MSH6 heterodimer) and Mutsb (MSH2/MSH3 heterodimer) recognise mismatched bases and recruit MutL (MLH1) to excise the mismatched bases. DNA polymerase fills the gaps
3) Recombination errors:
NAHR - misalignment of homologous mediated by LCRs can result in deletions/duplication
name 4 mechanisms of DNA repair & associated disorders
1) Mismatch Repair (MMR):
- MSH2/MSH3 heterodimer) recognise mismatched bases and recruit MutL (MLH1) to excise the mismatched bases. DNA polymerase fills the gaps
- MSH2/MSH6/MLH1 associated with Lynch syndrome (HNPCC)
2) BER - base excision repair:
- removal of oxidative damage / damaged bases
- MUTYH mutations ass. with colon cancer
3) NER - nucleotide excision repair:
- removal of pyrimidine dimers (UV radiation)
- Xeroderma Pigmentosa (XP) & Cockayne Syndrome
4) dsDNA breaks (DSB):
- via Homologous recombination repair, which uses WT stand from homologue as template for repair.
- requires RAD51 part of the BRCA1/BRCA2 pathway
Provide 2 examples of disorders associated with pseudogenes
1) SMA (spinal muscular atrophy)
- AR disorder
- due to deletions or gene conversions (in 98%) of SMN1 gene. (2% due to SNVs in SMN1)
- SMN2 pseudogene exists, shares 98% homology with SMN1
- key difference is an ex7 variant which disrupts a putative ESE (exonic splice enhancer) resulting in aberrant SMN2 splicing.
- SMN2 does have partial function and so increased copies of SMN2 can ameliorate the impact of SMN1 lose
2) CAH - congenital adrenal hypoplasia:
- AR
- due to deletions / gene conversions / mutations in geees in the cortisol from cholesterol pathway - notably CYP21a2
- pseudogene CYP21A1P - 98% homology, but harbours many deleterious inactivating variants
- region the gene & pseudogene reside prone to recombination
- results in deletions or gene conversions (which introduces those pathogenic variants into the coding gene)
- can result in virilasation in females and lethal salt wasting
What genes are tested as part of ‘floppy baby’ pathway
SMA, DM1, PWS
list 4 erros which may occur during meiosis I or II which result in aneuploidy
1) Recombination failure - achiasmatic non-disjunction
homologues separate to same pole
2) Premature homologue seperation - loss of cohesion be sister chromatids (MI)
3) Premature Sister chromatid separation - loss of cohesion between sister chromatids
4) Anaphase lag - failure of chromosome to attach to spindle apparatus and so fails to be pulled to poles (lags behind) and can be lost outside re-forming nucleus
What are the 4 possible outcomes of a pachytene cross:
1) Alternative:
Alternative (diagonal opposites) segregate - forms NORMAL & BALANCED gametes
2) Adjacent 1:
non-homologous centromeres travel together = UNBALANCED
3) Adjacent 2:
homologous centromeres travel together = UNBALANCED
4) 3:1
tertiary trisomy, 2 normal, 1 derivative
What 2 techniques can be used to investigate X-inactivation
1) Cytogenetically - Replication banding:
- treat cells initially with thymidine, following by Brdu, so that late replicating (inactive X) incorporates BrdU which stains late under Giesma
2) Molecular - HUMARA assay:
- based upon PCR amplification of CAG repeats in the AR gene
- genomic DNA is digested by methylation specific DNA (cuts unmethylated DNA - so only the active, unmethylated X is cut)
- PCR primers designed to flank RE digest sites - so if digested, no PCR product
- compared digested Vs undigested PCR products in normal Vs Skewed control.
Provide 2 different sizing strategies
1) Electrophoresis (gel, polyaccridimide, capillary)
2) PCR based amplification (LR-PCR; TP-PCR)
What are the types of enrichment available for NGS. Given examples
1) PCR based.
- can be in house LR-PCR based amplification
- or a commercial multiple kits - TruSeq Nextera
2) Enrichment based.
- use RNA / cDNA oligo probes as bait. Design baits to ‘tile’ over a region of interest
- genomic DNA will need fragmenting (RE digest or sonication)
- examples: SureSelect, Haloplex
Provide 2 examples of 3rd generation sequencing technologies
1) SMRT - single molecular real time sequencing (PacBio)
- real time sequencing
- long DNA strands run through a polymerase physically tethered to bottom of a specialism nanowell
- fluorescently labelled dNTPs added - and light emitted upon incorporation by DNA polymerase
- detection achieved due to miniscule size of well - so small that laser light cannot pass entirely through - but stops at point polymerase is tethered
- slides have a lawn of these wells - thousands of sequencings reactions occurring simultaneously
- PROs: fast; long reads (CNVs, indels, inversions etc)
2) Nanopore:
- DNA strand passes through nanopore
- unique electrical signature of each base is measured as passes through pore
- PROs: very quick; hand held device
- CONs: high error rate
