week 3

Question 1

Process of Next gen sequencing 5 steps

Answer 1

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

Question 2

What is PCR used for

Answer 2

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

Question 3

Process of PCR 4 steps

Answer 3

98 to denature

47-72 to allow primers to anneal

68-72 to allow extension

Repeat getting exponential amplification

Question 4

what is in solution of PCR

Answer 4

DNA, primers, free nucleotides, buffer

Question 5

sanger Variants detected

Answer 5

Unknown mutationsincluding SNVsand small duplications

Question 6

Sanger method 4 steps

Answer 6

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

Question 7

size Sanger can go up to

Answer 7

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

Question 8

Time to complete sanger

Answer 8

2-3 days

Question 9

Pros of sanger- 2

Answer 9

variety of mutations detected, no special equipment needed

Question 10

cons of sanger-3

Answer 10

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

Question 11

What is FISH

Answer 11

flourescence in situ hybridization

Question 12

variants detected in FISH

Answer 12

Targetedgenecopy numberchanges and targeted SVs

Question 13

Method of FISH 2 steps

Answer 13

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

Fluorescence microscopy is used for detection

Question 14

time to complete FISH

Answer 14

2-3 days

Question 15

Pros of FISH

Answer 15

easly detectsgenecopy numberchanges and targeted SVs

Question 16

cons of FISH

Answer 16

Requires paraffin-embedded tissue on unstained slides

Question 17

Variants dectect in Exome sequencing

Answer 17

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

Question 18

Method of exome sequencing

Answer 18

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

Question 19

time to complete exome sequencing

Answer 19

several days to weeks

Question 20

pros of exome sequencing

Answer 20

enables simultaneous detection of many variations in single assay

Question 21

cons of exome sequencing

Answer 21

expensive

Question 22

Variants detected by whole genome sequencing

Answer 22

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

Question 23

method of whole genome sequencing 3 steps

Answer 23

Random shearing of genomicDNAmolecules

NGS and specialized bioinformatics analysis.

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

Question 24

time to complete whole genome sequencing

Answer 24

several days to weeks

Question 25

pros of whole genome sequencing

Answer 25

Most comprehensive | Enables simultaneous detection of many variations

Question 26

cons of whole genome sequencing

Answer 26

expensive low throughput

Question 27

Targeted gene analysing panels variants tested

Answer 27

specific mutations in given sample, SNPS, INDELs

Question 28

method of Targeted gene analzing panels

Answer 28

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

Question 29

pros of Targeted gene analzing panels

Answer 29

Most cost effective

Question 30

what is MPLA

Answer 30

multiplex ligation dependent probe amplification

Question 31

variants tested by MPLA

Answer 31

Exon and gene copy number, typically whole exon dels or dups

Question 32

Method of MPLA 4 steps

Answer 32

primers that hybridise to patient DNA. bound primers are enzymatically ligated ligation products are amplified using multiplex PCR. Amplified products are detected and analysed by capillary electrophoresis.

Question 33

time to complete MPLA

Answer 33

2-3 days

Question 34

pros of MPLA

Answer 34

allows amplification of multiple targets with only single primer pair Quick able to detect multiple mutations simultaneously. No special equipment required. Can detect targeted SNVs at 10%

Question 35

cons of MPLA

Answer 35

Both targeted SNV and exon and gene copy number variants are specific

Question 36

SNP array method 4 steps

Answer 36

DNA amplified digested and labelled put onto array, hybridised and compare with non patient DNA to see differences

Question 37

Pros of SNP array

Answer 37

high throughput of thousands of SNPs in single experiment

Question 38

Taqman variants detected

Answer 38

SNP

Question 39

method of taqman

Answer 39

two primers probe for each SNP. Polymerisation to strand strand displaces reporter and reporter is cleaved and quencher removed so polymerisation then completed

Question 40

Pros of taqman

Answer 40

fast and simple

Question 41

Variants detected by BEAMing

Answer 41

``` Targeted known mutations including SNVs small insertions deletions,  indels rearrangements. ```

Question 42

method of BEAMing 5 steps

Answer 42

Circulating cell free DNA (cfDNA), containing ctDNA, is extracted from the blood plasma The gene of interest is pre-amplified using PCR The amplified DNA is mixed with beads coated with primers to capture the tag sequences and ePCR is performed, fluorescent probes to mutations of interest are hybridized to the amplified DNA Magnetic flow recovery methods can be used for detection

Question 43

time to complete BEAMing

Answer 43

2-3 days

Question 44

pros of BEAMing

Answer 44

high sensitivity and specificity

Question 45

cons of BEAMing

Answer 45

only detect knwon