Interview

Question 1

Outline Sanger sequencing

Answer 1

Separate the strands, incorporate modified nucleotides which terminate the process when added. Fragments ran on gel as per size. Each nucleotide is differently labelled so can tell which nucleotide has been incorporated.

Question 2

What are the types of whole genome sequencing?

Outline each

Answer 2

Illumina
Genome cut into 100-150bp fragments, amplified by PCR, add all copies to own spot and flood each spot with nucleotides and DNA polymerase. Each nucleotide has its fluorescent label. Terminator added after each incorporation. Terminator removed and process repeated.

454
Fragments up to 1Kb, multiple PCR copies of each fragment is attached to each bead which is attached to a well, well flooded with nucleotides one at time and then washed away, then the next nucleotide is added, Produces graph showing which nucleotide has been incorporated.

Proton/PGM
Same as 454 but with 200bp fragments, adds 1 nucleotide at a time but measures pH change caused if a nucleotide is added.

Question 3

What is karyotyping?

Answer 3

Number and appearance of chromosome in eukaryotic nucleus

Question 4

What is aneuploidy?

Answer 4

Presence of abnormal chromosome number

Question 5

What are the types of banding?

Answer 5

g-banding
Giesma stain following trypsin digestion. Dark and light bands. Dark=heterochromatin, late replicating and AT rich
Light=euchromatin, early replicating and GC rich

R banding
Reverse of G banding

c Banding
Giesma staining of constitutive heterochromatin (centromeres and telomeres)

q banding
Fluorescent dyes (quinacrine), similar stain to G banding but yellow, binds AT rich regions and hence heterochromatin more stained 

T banding
Telomeres stained

Question 6

What does banding show?

Answer 6

Visualisation of the karyotypes

Question 7

What is FISH used for?

How does it work?

Answer 7

Search for specific DNA sequences in chromosomes
Probes with flourescent tags for specific DNA sequence added to samples. DNA dyed with DAPI for visualisation. Flourescent microscope used to visualise where the probes have bound.

Question 8

What is comparative genomic hybridisation?

Answer 8

Used for analysing copy number variations. Only useful for unbalanced chromosomal abnormalities which will affect copy number. 5-10 mega base resolution, better than FISH or giesma banding. Compares sample to reference, female is preferred as have two X chromosome.
Nick translation used to label each DNA sample with modified nucleotides, either labelled with fluorophore or biotin which is target by conjugated enzymes. Areas of repeats are blocked. Ratio of sample and reference tag used to calculate if any deletions or insertions have occurred.

Question 9

What is array comparative genomic hybridisation?

Answer 9

Version of CGH which cuts the DNA into 100kb fragments. Fragments of sample and reference have own spot on micro array. Can find location of abnormalities using fragmentation pattern.

Question 10

What are single nucleotide polymorphisms?

Answer 10

variation in single nucleotide which occurs at a applicable degree within the population, such as sickle cell anaemia

Question 11

Outline a SNP array

Answer 11

DNA fragmented and a fluorescent tag is added. allele specific oligonucleotide probes on the array hybridise with sequences if the SNP is present. Detection system then searches for florescence.

Question 12

What is the genetic cause of down syndrome?

Answer 12

Trisomy 21
Gain of a third chromosome 21, 95% cases, or translocation of long arm of 21 to another chromosome, usually 14. Parents may have the translocation with no symptoms but more likely to pass on extra 21 material; 15% is mother and 5% is father.

Question 13

What is the genetic cause of klinefelter syndrome?

Answer 13

Male with two X chromosomes, leads to infertility. Due to sex chromosomes not separating during gamete formation; XX egg or XY sperm leads to XXY foetus

Question 14

What is the genetic cause of Patau syndrome?

Answer 14

Trisomy 13

Gain of third chromosome 13 or long arm of chromosome 13 to another chromosome

Question 15

What is the genetic cause of Cri du chat?

Answer 15

Loss of end of short arm of chromosome 5

Question 16

What is the genetic cause of Edwards syndrome?

Answer 16

Trisomy 18

Extra copy of chromosome 18, two copies in a gamete. Or translocation of long arm of 18 to another chromosome

Question 17

What is the genetic cause of 18q-?

Answer 17

Deletion of between 3 and 30,000 Mb from the long arm of chromosome 18

Question 18

What is the genetic cause of Turner syndrome?

Answer 18

Loss of all, or part of, one X chromosome. Monosomy, loss of a X chromosome, partial monosomy, loss of p arm of one X chromosome or isochromosome, an X chromosome with two q arms

Question 19

What is the genetic cause of Cystic Fibrosis?

Answer 19

Most commonly due to deletion of three nucleotides from q arm of chromosome 7. Causes loss of F508.

Question 20

What is the genetic cause of Sickle Cell Anaemia?

Answer 20

Single A to T mutation on chromosome 11. Having both alleles gives disease, having only one makes a carrier.

Question 21

What is the genetic cause of Angelman syndrome?

Answer 21

Loss of full complement from chromosome 15 received from maternal gamete. 4 Mb deletion UBE3A gene.