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Pharmacogenomics > week 3 > Flashcards

week 3 Flashcards

1
Q

Process of Next gen sequencing 5 steps

A

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

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2
Q

What is PCR used for

A

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

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3
Q

Process of PCR 4 steps

A

98 to denature

47-72 to allow primers to anneal

68-72 to allow extension

Repeat getting exponential amplification

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4
Q

what is in solution of PCR

A

DNA, primers, free nucleotides, buffer

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5
Q

sanger Variants detected

A

Unknown mutationsincluding SNVsand small duplications

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6
Q

Sanger method 4 steps

A

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

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7
Q

size Sanger can go up to

A

The reaction primers can be extended by up to about 1,000 nucleotides,

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8
Q

Time to complete sanger

A

2-3 days

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9
Q

Pros of sanger- 2

A

variety of mutations detected, no special equipment needed

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10
Q

cons of sanger-3

A

Labor intensive
requires mutantDNAto be present at 20–25%-
cannot detect changes in exon

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11
Q

What is FISH

A

flourescence in situ hybridization

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12
Q

variants detected in FISH

A

Targetedgenecopy numberchanges and targeted SVs

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13
Q

Method of FISH 2 steps

A

Fluorescent probes are used to locategenesor sequences of interest on one or morechromosome

Fluorescence microscopy is used for detection

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14
Q

time to complete FISH

A

2-3 days

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15
Q

Pros of FISH

A

easly detectsgenecopy numberchanges and targeted SVs

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16
Q

cons of FISH

A

Requires paraffin-embedded tissue on unstained slides

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17
Q

Variants dectect in Exome sequencing

A

Substitutions, duplications, insertions, deletions, indels, andgenecopy numberchanges.

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18
Q

Method of exome sequencing

A

Hybridization capture of regions of interest,
NGS, GenomicDNAfragments are hybridized in solution to sequence-specific capture probes for all protein-coding exons throughout thegenome

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19
Q

time to complete exome sequencing

A

several days to weeks

20
Q

pros of exome sequencing

A

enables simultaneous detection of many variations in single assay

21
Q

cons of exome sequencing

A

expensive

22
Q

Variants detected by whole genome sequencing

A

Substitutions, duplications, insertions, deletions, indels,genecopy numberchanges, andchromosomeinversions and translocations

23
Q

method of whole genome sequencing 3 steps

A

Random shearing of genomicDNAmolecules

NGS and specialized bioinformatics analysis.

These tests interrogate both coding and non-coding regions of the humangenome.

24
Q

time to complete whole genome sequencing

A

several days to weeks

25
Q

pros of whole genome sequencing

A

Most comprehensive

Enables simultaneous detection of many variations

26
Q

cons of whole genome sequencing

A

expensive

low throughput

27
Q

Targeted gene analysing panels variants tested

A

specific mutations in given sample, SNPS, INDELs

28
Q

method of Targeted gene analzing panels

A

panels contain select set of genes or regions with known or suspected association with disease or phenotype
can be purchased or custom designed

29
Q

pros of Targeted gene analzing panels

A

Most cost effective

30
Q

what is MPLA

A

multiplex ligation dependent probe amplification

31
Q

variants tested by MPLA

A

Exon andgenecopy number, typically whole exon dels or dups

32
Q

Method of MPLA 4 steps

A

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.

33
Q

time to complete MPLA

A

2-3 days

34
Q

pros of MPLA

A

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%

35
Q

cons of MPLA

A

Both targeted SNV and exon andgenecopy numbervariants are specific

36
Q

SNP array method 4 steps

A

DNA amplified

digested and labelled

put onto array,

hybridised and compare with non patient DNA to see differences

37
Q

Pros of SNP array

A

high throughput of thousands of SNPs in single experiment

38
Q

Taqman variants detected

A

SNP

39
Q

method of taqman

A

two primers probe for each SNP.

Polymerisation to strand

strand displaces reporter and reporter is cleaved and quencher removed so
polymerisation then completed

40
Q

Pros of taqman

A

fast and simple

41
Q

Variants detected by BEAMing

A 
Targeted knownmutations
including SNVs
small insertions
deletions, 
indels
 rearrangements.
42
Q

method of BEAMing 5 steps

A

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

43
Q

time to complete BEAMing

A

2-3 days

44
Q

pros of BEAMing

A

high sensitivity and specificity

45
Q

cons of BEAMing

A

only detect knwon

Pharmacogenomics (11 decks)

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