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(6-10) Translational Medical Science > Genetic testing > Flashcards

Flashcards in Genetic testing Deck (58)
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1
Q

two main types of genetic testing

A

molecular testing

cytogenetics

2
Q

molecular testing

A

sequencing of specific genes or genotyping specific small mutations based on phenotype

3
Q

what does molecular testing use

A

PCR and sequencing

4
Q

types of molecular testing

A

whole genome sequencing

whole exome sequencing

5
Q

whole genome sequencing

A

the process of determining the complete DNA sequence of an individual at one time

6
Q

process of WGS

A

1) obtain DNA sample
2) purify DNA and break into short fragments
3) sequence many times over

7
Q

why sequence the whole genome

A

to find the cause of an unexplained disease that appears to be genetic

8
Q

Whole exome sequencing

A

sequencing of all exomes in the genome

9
Q

exomes

A

expressed part of the genome and some regulatory sequences

10
Q

how is the exome filtered out of the exome

A

DNA gets fragmented and passed through a filter which traps the coding sequences (complimentary binds with exome ssDNA)

11
Q

the exome genome is compared to

A

a start human reference genome sequences

12
Q

what can be found in WES

A

presence of known SNP polymorphism

rare differences

unique differences

INDELS

13
Q

why sequence an exome

A

cheaper than sequencing the whole genome

- get almost as much useful info as WGS

14
Q

What is PCR sued to do

A

amplify specific segments of DNA

15
Q

why is PCR important

A

amplification allows detection of pathogenic virus’ and bacteria, as well as identification of individuals e.g. in crime

16
Q

what ingredients are needed for PCR

A

1) primers
2) dNTPs (free nucleotides)
3) Taq polymerase
4) DNA template to be copied
5) buffer

17
Q

what is used in PCR

A

a thermocycler

18
Q

process of PCR

A

1) denaturing (90-95)- separate double strands
2) annealing (50-56)- temp lowered to enable primers to bind
3) extension (72)- temp raised and new strands synthesised by polymerase

–> cycle repeated- DNA doubles everytime

19
Q

how is whole genome sequence glad whole exome sequencing carried out

A

using NGS

20
Q

what was first generation sequencing

A

Sanger

21
Q

outline the sanger method

A
  • DNA to be sequenced is amplified (PCR)
  • DNA is then denatured using heat– ssDNA
  • DNA split into template and complimentary strand
  • primer for specific region of DNA is then annealed to the template strand to allow addition of nucleotides
  • 4 reaction mixtures are set up
  • in each mixture the template strand of DNA, DNA polymerase and free nucleotides (dNTP are added)
  • modified ddNTPs (tagged with fluorescence) are then added to the mixture (one type of fluorescent A, T, C ,G added to each well)
  • ddNTPs cause termination of polymerisation due to lacking a hydroxyl group
  • PCR occurs- by the end each mixture will yield PCR products of differing lengths due to replication being randomly terminated
  • DNA then separated on the basis of six on a gel electrophoresis page
  • can detect order when an x ray is used due to fluorescent bands that appear
22
Q

next generation sequences method is also called

A

second generation

23
Q

why is NGS better than first generation (sanger)

A
  • more accurate
  • quicker
  • reduced manpower
  • reduced cost
24
Q

name a type of NGS

A

e.g. pyrosequencing

25
Q

name 3 cytogenetic testing methods

A
  • karyotyping
  • FISH
  • microarrays
26
Q

karyotyping is a

A

cytogenetic test

27
Q

karyotyping is the process of

A

ordering and pairing all chromosomes of an organism

28
Q

chromosome 1 is the

A

largest chromosome

29
Q

chromosome 22

A

is the smallest

30
Q

karyotyping is a test to…

A

identity the size, shape and number of chromosomes in a sample of body cells

31
Q

how many chromosomes in body cell

A

46

32
Q

autosomes

A

Chr1-22

–> 44 autosomes

33
Q

karyotyping can be used to detect

A
  • abnormal numbers of chromosomes

- abnormal size e.g. CNVs

34
Q

name two karyotyping methods

A
  • light microscope

- G-banded karyotypes

35
Q

light microscope: karyotyping

A

can detected abnormal number of chromosomes

36
Q

can detected abnormal number of chromosomes

A

aneuploidy/ polyploidy

37
Q

G-BANDED KARYOTYPE

A

shows structural problems with chromosome

38
Q

what stain is used in G-banded karyotype

A

Giesma stain

  • stains condensed chromosomes
  • causes dark bands
39
Q

Gismo stains which part of the chromosomes

A

AT rich regions– can show homologous chromosomes e.g. will have bands in the same place

40
Q

microarray is another type of

A

cytogenetic test

41
Q

microarrays are a

A

tool used to detect the expression of thousands of genes at the same time

42
Q

microarray process

A

1) mRNA extracted from reference (healthy) and experimental sample (patient)
2) mRNA is reverse transcribed to cDNA
3) reference cDNA is tagged green
4) experimental cDNA is tagged red
5) both cDNA samples are combined and added to a microarray chip
6) cDNA binds to its complimentary base pair which is attached to the chip
7) chip is rinsed
8) chip put through a laser scanner (which activates fluorescent dye)
9) indicates which evens are being expressed (if they ar being expressed differently)

43
Q

if yellow

A

both genomes express the same gene

44
Q

name two types of microarray

A

SNP-array

Array- CGH

45
Q

SNP- array detect

A

single nucleotide polymorphisms (most common type of mutation)

46
Q

Array-CGH

A

comparative genomic hybridisation

47
Q

Array-CGH analyses

A

CNVs

48
Q

CNV

A

copy number variants

49
Q

FISH is a type of

A

cytogenetic test

50
Q

FISH stands for

A

fluorescence in situ hybridisation

51
Q

FISh was developed to

A

detect and locate presence or absence of specific DNA sequence on the chromosome

52
Q

aim of FISH

A

to detect abnormal chromosomes- caused by the binding of fluorescent probe that is complimentary to a specific region on a chromosome
- shows if chromosome location is mutated or not

53
Q

what are standard diagnosis tests

A
  • microarrays

- molecular testing for specific conditions

54
Q

targeted next regeneration sequencing

A

gene panels based on phenotypes

55
Q

genome wide sequencing using NGS

A
  • whole exome sequencing

- whole genome sequencing

56
Q

how many protein coding genes

A

20,000

57
Q

what else can be sequences

A

the clinical exome

58
Q

the clinical exome

A

includes are 4,000 known disease genes