GiM: Techniques of Chromosomal Analysis

Question 1

What are some analysis techniques that look at the whole genome?

Answer 1

• G banding
• Next Generation Sequencing
• Microarrays

Question 2

What are some analysis techniques that are more targeted?

Answer 2

• FISH
• MLPA
• QF-PCR or qPCR

Question 3

What are some applications of FISH?

Answer 3

Copy Number imbalance
Aneuploidy
Confirmation of G-banding

Question 4

3 types of FISH probe?

Answer 4

• Unique sequence (can light up any region of DNA that interested in, like deletions and duplications)
• Centromeric (can tell how many XX and XY)
• Paints

Question 5

What is MLPA?

Answer 5

Multiplex ligation dependent probe amplification (alternative to FISH)
Fragment analysis: peak area= amount of target sequence (normal: 1.0 duplication: 1.35 deletion: 0.65)

Question 6

What is array-CGH?

Answer 6

Microarray comparative genomic hybridisation (for Genome-wide screening)
Replacing karyotyping as 1st line test.

Question 7

Requirements of array-CGH?

Answer 7

3ml blood in EDTA

Control DNA from same sex

Question 8

Advantages and disadvantages of array-CGH?

Answer 8

Advantages:
-Early Diagnosis (1st line test)
-High resolution (increased diagnostic hit rate)
-Greater accuracy of location/size of imbalances
Disadvantages:
-For dosage changes only (not balanced rearrangements or mutations)
-Low level mosaics not detected
-Needs good-quality DNA

Question 9

What is Quantitative fluorescent PCR (QF-PCR)?

Answer 9

PCR amplification of short term repeats (STRs) using fluorescent primers.
Products visualised and quantified as peak areas using an automated DNA sequencer.

Question 10

What can QF-PCR be used for?

Answer 10

• Prenatal aneuploidy detection (aneuploidy= >2 markers with abnormal dosage) Size of product is directly related to number of ATTT repeats.
• Identify prenatal trisomies (if 3 peaks or 2:1 ratio is indicative)

Question 11

What are the 3 types of sample that can be used for G-banding?

Answer 11

1) Blood= 2-5ml unclotted (with heparin). Stimulated T-lymphocytes, culture 2-3 days.
2) Amniotic Fluid= separate cells from portion and culture for 7-14 days.
3) Chorionic Villi= separate maternal from foetal tissue then QF-PCR, then culture for 7-14 days.

Question 12

What are 3 types of prenatal diagnosis?

Answer 12

Amniocentesis (16wks)
Chorionic villus biopsy (CVS, 12wks)
NIPT (non invasive- maternal blood sample, extract circulating free foetal DNA) (12wks)

Question 13

What are 5 biochemical markers and how are they linked to a Downs Syndrome risk?

Answer 13

• PAPP-A (produced by placental syncytiotrophoblasts): levels reduced in Downs
• Beta-hCG : levels raised in Downs
• AFP: levels reduced in Downs
• uE3: levels reduced in Downs
• Inhibin-A: levels raised in Downs

Question 14

What is the process of G-banding?

Answer 14

1) The metaphase chromosomes are treated with trypsin (to partially digest) and then stained with Giamsa stain.
2) Heterochromatic regions (AT-rich and relatively gene-poor) stain more darkly.
3) Less dense chromatin (GC-rich and more transcriptionally active) appear as lighter bands.