L10&11- Techniques of chromosomal analysis

Question 1

Whole genome test types

Answer 1

G-banding
Next ten sequencing
Microarrays

Question 2

Targeted testing

Answer 2

FISH
MLPA
QF-PCR/qPCR

Question 3

Process of G banding

Answer 3

Cell culture –> Mitotic arrest –> Hypotonic –>Fixation –>Trypsin & Leishman’s stain –>Banding - AT and GC rich regions

Question 4

Molecular cytogenetic techniques

Answer 4

Fluorescent in situ hybridisation (FISH)

Multiplex ligation dependent probe amplification (MLPA)

Microarray comparative genomic hybridisation (array CGH)

Next generation sequencing

Quantitative fluorescent PCR (QF-PCR)

qPCR

Question 5

Fluorescent in situ hybridisation (FISH)

Answer 5

Detection of DNA material on slides using fluorescent

Dyes & UV light

Question 6

FISH process

Answer 6

Labelling (fluorochrome) the probe (which will bind to the target you are looking for e.g chromosome 18 or X/Y) –> denaturation –> mix in with the target (sample) —> hybridisation –> (post hybridisation washing) –> Visualisation (24 hours) with UV light

Question 7

Types of FISH probes

Answer 7

• unique sequence (specific place on an arm?)
• Centromeric (middle of the chromosome will light up)
• Paints (whole chromosome lights/painted up!)

Question 8

Applications of FISH

Answer 8

Copy number imbalance

Aneuploidy

Confirmation/ clarification of G-banding

Confirmation of array CGH

Identifying specific abnormalities in cancer

Question 9

Copy number variability

Answer 9

A copy number variation (CNV) is when the number of copies of a particular gene varies

For example, the chromosome that normally has sections in order as A-B-C-D might instead have sections A-B-C-C-D (a duplication of “C”) or A-B-D (a deletion of “C”)

One gene or contiguous genes
Pathogenic or benign

Question 10

Molecular cytogenetic methods to assess copy number

Answer 10

FISH

MLPA

Microarray CGH

Next generation sequencing

QF-PCR

qPCR

Question 11

MLPA

Answer 11

Multiplex Ligation-dependent Probe Amplification

DNA-based

Multiplex PCR

Copy no. changes in up to 50 different genomic locations simultaneously (allows multiple targets to be amplified)

Alternative to FISH

Question 12

MLPA process

Answer 12

You have two primers (X and Y one of which has a stuffer sequence) –> added to the DNA sample and allowed to hybridise –> ligation reaction occurs –> the two parts of the primers (two parts of hybridised probe) are ligated by a thermostable ligase –> PCR amplification using a single primer pair

(More in detail regarding probes) The probes are hybridized against the target DNA and subsequently ligated. Only if ligation happened, a functional PCR strand appears, so that amplification only happens if target DNA is present in the sample. The amount of PCR product is proportional to the amount of target DNA present in the sample, making the technique suitable for quantitative measurements

Question 13

MicroArray CGH (comparative genomic hybridisation)

Answer 13

Genome-wide screen

Hybridise sample & control DNA to a microarray “chip” 1000s of DNA spots (oligonucleotides)

Genomic imbalances (copy number variants) at high resolution (10-10000x conventional cytogenetics)

Increased detection rates

Replacing karyotyping as 1st line test

Question 14

What are the requirements for MicroArray CGH?

Answer 14

3ml blood in EDTA (also 1-2ml lithium heparin blood for cell culture if follow up studies needed)

Control DNA from same sex

Question 15

MicroArray CGH measures?

Answer 15

Copy number variability…..mostly imbalances

At least 3 oligonucleotides required for any call

Call imbalances >150000 bases, ie 150 Kb

Database searches to ascertain pathogenicity of imbalances

Question 16

Advantages of array CGH

Answer 16

Early diagnosis -1st line test, reduces need for other tests and avoids the “diagnostic odyssey”

High resolution = increased diagnostic hit rate

Greater accuracy of location/size of imbalances

Information on relevant genes

Question 17

Disadvantages of array CGH

Answer 17

Dosage changes only (Gene dosage is the number of copies of a particular gene present in a genome) – not balanced rearrangements or mutations (usually only detects unbalanced chromosomal abnormalities)

Low level mosaics not detected

Non-pathogenic & uncertain pathogenic changes detected

Needs good quality DNA

Question 18

What do Next gen sequencing data analysis mean

Answer 18

Increased test:control ratio = gain

Decreased test:control ratio = deletion

Question 19

Quantitative fluorescent PCR (QF-PCR)

Answer 19

PCR amplification of short tandem repeats (STRs e.g Microsatellite tetranucleotide repeat marker) [chromosome-specific, repeated DNA sequences] using fluorescent primers

Products visualised & quantified as peak areas using an automated DNA sequencer

PCR of tetranucleotide repeat analysed on a fluorescent sequencer

Question 20

Prenatal aneuploidy detection

Answer 20

Quantitative fluorescent PCR (QF-PCR)

DNA extraction from amniotic fluid or chorionc villi
PCR amplification – primers from chromosomes 13, 18, 21, X and Y
DNA dosage in up to 4-5 markers/chromosome
aneuploidy =>2 markers with abnormal dosage

Question 21

qPCR (real time PCR)

Answer 21

Quantitative comparison vs reference gene & normal control patient (amplify & quantify)

Confirming small CNVs

When FISH unsuitable

Primer design

Question 22

qPCR quantification

Answer 22

Relative Quantitation (RQ) - compares difference in concentration between patient sample & normal control assessed by 2 different primer sets

RQ value is expressed as a ratio relative to 1 - a deletion has an expected value of 0.5 & a duplication an expected value of 1.5

Question 23

What would qPCR show for trisomy 21?

Answer 23

Trisomic 2:1 ratio

Question 24

Robertsonian translocation

Answer 24

rare form of chromosomal rearrangement that, in humans, occurs in the five acrocentric chromosome pairs, namely 13, 14, 15, 21, and 22

Question 25

Look more on robertsonian translocation in lectures 10&11

Answer 25

slides 50 and on

Question 26

Robertsonian chain trivalent

Answer 26

formation of a Robertsonian translocation involving chromosomes 14 and 21, and the formation of a trivalent in meiosis

Question 27

Prenatal diagnosis procedures

Answer 27

1. Amniocentesis (16w) –> QF PCR –> G banding
2. Chorionic villus biopsy (CVS, 12w) –>QF PCR –> G banding
3. NIPT (12w) -Non-Invasive Prenatal Testing

Question 28

Non-invasive prenatal testing (NIPT)

Answer 28

Maternal blood sample

Extract circulating free fetal DNA

Assess aneuploidy of 13, 18, 21 (NGS)

Risk for aneuploidy – invasive test to confirm

Reduces no. of invasive tests

Question 29

Array CGH and prenatal diagnosis

Answer 29

Replacement of cell culture if abnormal scan
Advantages
Increased resolution
Higher detection rate
Disadvantages
Ethical, eg small duplication & associated autism
Need parental follow up

Question 30

Translocation in leukaemiaEg t(9;22) in CML (chronic myeloid leukaemia)

Answer 30

Philadelphia chromosome - is a specific genetic abnormality in chromosome 22 of leukemia cancer cells

This chromosome is defective and unusually short because of reciprocal translocation of genetic material between chromosome 9 and chromosome 22, and contains a fusion gene called BCR-ABL1.