Tests

Question 1

steps of G-banding (giemsa)

Answer 1

1. extract lymphocyte and culture at 37 deg for 3 days
2. add colchicine to arrest in metaphase
3. add hypotonic saline to cause swelling
4. lyse cells, mount on slide and giemsa stain

Question 2

what kind of chromosome abnormalities does g-banding identify

Answer 2

big ones

• chromosome number
• translocations
• large deletion/duplications

Question 3

steps of FISH (fluorescent in situ hybridisation)

Answer 3

1. all the same as g-banding
2. denature DNA(make it single stranded)
3. add fluorescent probe (complementary)

Question 4

what abnormalities does FISH detect

Answer 4

large deletions/duplications:

• prader willi
• 22q11 del
• cri-du-chat

Question 5

what is quantitate fluorescence PCR (QF-PCR)

Answer 5

uses microsatellites to identify short tandem repeats(STRs):

• no of repeats is specific to individual
• in trisomies- 3 copies

Question 6

what abnormality does QF-PCR tell us

Answer 6

tells us chromosome number

Question 7

what is array comparative genomic hybridisation (array-CGH)

Answer 7

1. patient and control DNA labelled with fluorescent dyes and applies to microassay (denatured)
2. both compete to anneal to probes on slide
3. should be equal binding, but if duplication or deletion then microassay is different

Question 8

what abnormalities does array-CGH show

Answer 8

microdeletions/duplications

Question 9

steps of sanger sequencing

Answer 9

1. strand seperation
2. anneal primer to 3’end
3. extension- dNTPs are added by Taq polymerase
4. termination - ddNTP(fluorescent nucleotide) is randomly added, terminating the complementary strand
5. denature new DNA, keeping new strand
6. load fragments onto gel and apply current
7. reference to normal DNA and detect point mutations

Question 10

what abnormalities does sanger sequencing detect

Answer 10

DNA level - substitutions/small indels

Question 11

what is next generation sequencing

Answer 11

sequence whole genome

Question 12

what abnormalities does nect generation sequencing identify

Answer 12

DNA level - substitutions/small indels

Question 13

which tests show big chromosome abnormalities

Answer 13

G-banding
FISH
QF-PCR

Question 14

what is the disadvantage of QF-PCR

Answer 14

you have to suspect a trisomy

Question 15

what is the disadvantage of sanger sequencing

Answer 15

you have to suspect a gene