week 3 Flashcards
Process of Next gen sequencing 5 steps
DNA must be broken into smaller sequences
split into two strands, denatured
Then hybridized to a surface, bead
Fragment then amplified by PCR
Left with cluster of DNA, then sequenced together with fluorescent nucleotides tat emit light of a certain colour so know what nucleotide is added
What is PCR used for
Used to amplify single copy of few copies of segment of DNA
Can be used to investigate size of a Region od DNA or DNA sequence
Process of PCR 4 steps
98 to denature
47-72 to allow primers to anneal
68-72 to allow extension
Repeat getting exponential amplification
what is in solution of PCR
DNA, primers, free nucleotides, buffer
sanger Variants detected
Unknown mutationsincluding SNVsand small duplications
Sanger method 4 steps
Sequencing primers hybridized to the PCR product
extended usingDNApolymerase, the four deoxynucleotides (dNTPs), and a mixture of fluorescently labeled dideoxynucleotides (ddNTPs)
Random incorporation- termination of strands at each location along the sequence.
Capillary electrophoresis separates the strands by size
size Sanger can go up to
The reaction primers can be extended by up to about 1,000 nucleotides,
Time to complete sanger
2-3 days
Pros of sanger- 2
variety of mutations detected, no special equipment needed
cons of sanger-3
Labor intensive
requires mutantDNAto be present at 20–25%-
cannot detect changes in exon
What is FISH
flourescence in situ hybridization
variants detected in FISH
Targetedgenecopy numberchanges and targeted SVs
Method of FISH 2 steps
Fluorescent probes are used to locategenesor sequences of interest on one or morechromosome
Fluorescence microscopy is used for detection
time to complete FISH
2-3 days
Pros of FISH
easly detectsgenecopy numberchanges and targeted SVs
cons of FISH
Requires paraffin-embedded tissue on unstained slides
Variants dectect in Exome sequencing
Substitutions, duplications, insertions, deletions, indels, andgenecopy numberchanges.
Method of exome sequencing
Hybridization capture of regions of interest,
NGS, GenomicDNAfragments are hybridized in solution to sequence-specific capture probes for all protein-coding exons throughout thegenome
time to complete exome sequencing
several days to weeks
pros of exome sequencing
enables simultaneous detection of many variations in single assay
cons of exome sequencing
expensive
Variants detected by whole genome sequencing
Substitutions, duplications, insertions, deletions, indels,genecopy numberchanges, andchromosomeinversions and translocations
method of whole genome sequencing 3 steps
Random shearing of genomicDNAmolecules
NGS and specialized bioinformatics analysis.
These tests interrogate both coding and non-coding regions of the humangenome.
time to complete whole genome sequencing
several days to weeks
pros of whole genome sequencing
Most comprehensive
Enables simultaneous detection of many variations
cons of whole genome sequencing
expensive
low throughput
Targeted gene analysing panels variants tested
specific mutations in given sample, SNPS, INDELs
method of Targeted gene analzing panels
panels contain select set of genes or regions with known or suspected association with disease or phenotype
can be purchased or custom designed
pros of Targeted gene analzing panels
Most cost effective
what is MPLA
multiplex ligation dependent probe amplification
variants tested by MPLA
Exon andgenecopy number, typically whole exon dels or dups
Method of MPLA 4 steps
primers that hybridise to patientDNA.
bound primers are enzymatically ligated
ligation products are amplified using multiplex PCR.
Amplified products are detected and analysed by capillary electrophoresis.
time to complete MPLA
2-3 days
pros of MPLA
allows amplification of multiple targets with only single primer pair Quick
able to detect multiplemutationssimultaneously.
No special equipment required.
Can detect targeted SNVs at 10%
cons of MPLA
Both targeted SNV and exon andgenecopy numbervariants are specific
SNP array method 4 steps
DNA amplified
digested and labelled
put onto array,
hybridised and compare with non patient DNA to see differences
Pros of SNP array
high throughput of thousands of SNPs in single experiment
Taqman variants detected
SNP
method of taqman
two primers probe for each SNP.
Polymerisation to strand
strand displaces reporter and reporter is cleaved and quencher removed so
polymerisation then completed
Pros of taqman
fast and simple
Variants detected by BEAMing
Targeted knownmutations including SNVs small insertions deletions, indels rearrangements.
method of BEAMing 5 steps
Circulating cell freeDNA(cfDNA), containing ctDNA, is extracted from the blood plasma
Thegeneof interest is pre-amplified using PCR
The amplifiedDNAis mixed with beads coated with primers to capture the tag sequences and ePCR is performed,
fluorescent probes tomutationsof interest are hybridized to the amplifiedDNA
Magnetic flow recovery methods can be used for detection
time to complete BEAMing
2-3 days
pros of BEAMing
high sensitivity and specificity
cons of BEAMing
only detect knwon
