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GENE AMPLIFICATION
Cancer Genomics:
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This led to further technological developments in DNA
sequencing methodologies and thus better sequencing of the entire cancer genome
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Next generation sequencing (NGS) is the catch-all term used to describe a number of
different modern sequencing technologies
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These recent technologies allow us to sequence DNA and RNA much more quickly
and cheaply than previous technologies, and as such have revolutionised the study
of genomics and molecular biology
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In regards to cancer genetics, NGS can be used for:
• Whole exome sequencing – a catalogue of cancer genes
• Whole genome sequencing – providing a catalogue of all cancer mutations
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It requires 3 key steps:
• PCR on solid support – amplification step
• Reversible terminator sequence
• Read lengths ca
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The input sample must be cleaved into short sections (or fragments)
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PCR is carried out to amplify these 150bp reads, creating a spot with many copies of
the same read
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A terminator is
also present so only one base is added at a time
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The slide is then prepared for the next cycle: the terminators are removed, allowing
the next base to be added, and the fluorescent signal removed, preventing the signal
from contaminating the next image
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The process is repeated, adding one nucleotide at a time and imaging in between
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Computers are then used to detect the base at each site in each image and these are
used to construct a sequence
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All of the sequence reads will be the same length
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The Cancer Genome Atlas (TCGA) is a comprehensive and coordinated effort to
accelerate our understanding of the molecular basis of cancer through the
application of genome analysis technologies
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It has identified 33 cancer types from 11,000 samples
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• Tumour subtypes – revolutionized how cancer is classified
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There are
therefore more mutations than the number of human genes: each gene is mutated
~3 times
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some
breast cancer types have few mutations due to different lifestyles, stage of the
disease, predispositions (i
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susceptibility)
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Many mutations are found within the coding portions of the genome, such as
introducing stop codons into the frame
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Not All Mutations Contribute to Cancer:
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a driver mutation could lead to cells overcoming the selective barrier
that causes apoptosis – “just right instability” – thus resulting in cell immortality
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Passenger mutations are mutations that have no effect on the neoplastic process
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Every cancer
sample has its own repertoire of mutations thus allowing personalised therapy
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There are some genes that are found in over 50% of some cancer types, i
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p53 in
breast cancer
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However, many cancer genes are still unknown
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Whole Genome Sequencing:
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The v-myc avian myelocytomatosis viral oncogene homolog is a transcription factor
with a role in cell cycle progression, apoptosis and cellular transformation by binding
to Max
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The myc oncogene is a heavily recurrent mutated cancer gene, particularly found in
lymphomas but also in solid tumours
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Cancer clonal evolution is driven by somatic alterations
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These mutations escape chemotherapy due to the
inadequate treatment or driver mutations in the founding clones
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Pattern 2: a minor sub-clone survives chemotherapy and other treatment, gains
mutations and expands at relapse
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