Lecture 1/2 - the complexity of cancer Flashcards
Circos plot
represent data sequence of whole cancer cell genomes
all lines, dots and colours show extent of change
Inter-chromosomal structural variations
parts of the chromosome stuck to different chromosomes - swapped around
Intra-chromosomal structure variations
normally separated parts in chromosomes come together or parts are swapped around within a single chromosome
Loss of heterozygosity/allelic imbalance
loss of heterozygous during proliferation/mitotic recombination
one is non-functional so loss of the functional allele is particularly important for tumour suppressors
Copy number variation
in porto-oncogenes, changes in expression
Name the progressive hierarchy in order in cancer genomes
Interchromosomal intrachromosomal structural variations loss of heterozygosity/allelic imbalance copy number variation single nucleotide variation
Cancer genome sequencing process
- Shear DNA to 100bps fragments
- chemically ligate to a known adaptor sequence
- chemically attached chimeric DNA to solid chip surface
- Introduce probes and anchor which are colour coded with fluorescent fluorophores
- DNA ligase forms bonds between probes to anchor if complementary - shape must match in order for them to catalyse the reaction
- raise temperature as temp it takes for probe to be removed determines length
- Using DNA ligase and melting temp to ensure only correct probe in place
Cancer genome sequencing can also…
can also investigate positions 1-5 in any probe set
can also use offset anchor in order to extend the sequencing so build up with smaller parts
only take one signal therefore must amplify it
Cancer genome sequencing must amplify the signals by…
take two anchors to form a circle
add DNA polymerase and many nucleotides
DNA polymerase continuously goes around circle repeatedly to make many copies to increase signal strength
Nanomoles
attached to amplifying circle array and use probes to determine sequences
Raising temperature in cancer genome sequencing means…
the temperature it takes to be removed determines length. If the wrong one binds the ligase will not catalyse the reaction so temperature increase will just get it to fall off.
Chromosomal instability (CIN)
the ability to maintain the correct number and gross composition of chromosomes, two types
Numerical CIN
aneuploidy, wrong number of chromosomes
Structural CIN
gross compositional failure in chromosomes
Philadelphia chromosome
structural CIN underpinning myeloblastic leukaemia
reciprocal translocation between ends of chromosomes 9 and 22
Philadelphia chromosome is…
the fusion of the BCR-ABL proteins to produce a non-natural protein which is unregulated and highly expressed
ABL
protein kinase involved in proliferation and cell cycle progression
drives WBC progenitor proliferation
Myc CIN
Myc is a porto-oncogene that is amplified in neuroblastomas
Colorectal cancer occurs when…
loss of the second APC allele in familial adenomatous polyposis
Loss of heterozygosity
Wnt signalling and APC loss
Wnt down regulation doesn’t occur, so B-catenin is always released, so cells migrating up the crypt doesn’t stop proliferating
GSK cannot induce B-catenin degradation
Microsatellite instabilità (MIN)
all single nucleotide variations occurred due to unpaired DNA damage
microsatellite
variable length dinucleotide repeats
Microsatellite mutations
these are hypermutable with mismatches occur. DNA repair cuts out the loop but does not fix properly so require realigning instead
Deamination
changes cysteine to uracil, so when DNA is replicated one stranded results in U-A pairing rather than C-G
Depurination
Adenine is removed so when DNA is replicated, an A-T nucleotide pair is deleted
Mutations in mismatch repair proteins precipitate MIN
MSH creates ring around mismatch site, nucleoside creates nick in mismatch vicinity, ring finds nick and chooses way to create ssDNA to replace mismatch, polymerase replicates this properly and follows template to fix it
HNPCC
aka Lynch syndrome, a MIN example
Loss of function alleles for MSH and loop
Driver mutations
viral oncogenes, drivers of carcinogenesis
Driver mutation experiments
take fragmented DNA and transfect normal mice fibroblasts, inject these into mice
if oncogenes are present then tumours are formed
