S3: Application of Molecular Biology to Medicine

Question 1

What is Cytogenetics and Molecular Genetics Laboratory important for?

Answer 1

• Chromosome analysis
• Pre and post natal testing (e.g. foetal blood, amniotic fluid)
• Adult testing (tissue, blood)

Question 2

What investigations can be done in an Cytogenetics and Molecular Genetics Laboratory?

Answer 2

• G banding
• FISH
• QF-PCR
• Array-CGH

Question 3

Examples of structural variation in genetics

Answer 3

• Chromosomal abnormalities
• Translocations
• Copy number variations (large deletions/duplications)

Question 4

Examples of numerical abnormalities

Answer 4

• Autosomal e.g. Trisomy 12,18,21

- Sex chromosomes e.g. XO, XXY, XYY

Question 5

What are the three features scientists use to identify chromosomes?

Answer 5

1. Size
2. Banding Pattern
3. Centromere position

Question 6

What are the 4 centromere position names chromosomes have?

Answer 6

• Metacentric (middle)
• Sub-metacentric
• Acrocentric
• Telocentric (not found in humans)

Question 7

Describe G- banding

Answer 7

It is a giema stain which bands chromosomes together. The chromosomes are taken from the metaphase part of the cell. It is used to see how the karyotype of the patient differs from the norm.

Question 8

What are the 2 different sorts of chromatin?

Answer 8

1. Euchromatin is a GC base rich, it is loosely packed because it is so active so this allows it to unwind easily.
2. Heterochromatin is AT rich and it is tightly packed because here the genes are inactive (i.e. not being transcribed)

Question 9

Why does chomosomes have banding pattern with G-stain?

Answer 9

There are banding patterns on chromosmes because of the different types of chromatin and the way it is arranged to form the chromosomes.
It stains darkly to the heterochromatin regions and the euchromatin regions are lighter.

Question 10

What does 11p15.5 mean?

Answer 10

11 = chromosome 11
p = small arm
15 (next one down would be 14 resolution)
.5 = higher resolution is split by a decimal.

Question 11

Method of G -stain

Answer 11

• Extract lymphocytes and culture them
• Use lymphocytes to get hold for chromsomes
• Arrest lymphocyte when it is in metaphase so the chromosomes are in the structure we want
• Add hypotonic saline to cause swelling and lyse the cell
• Wash and mount chromosome on slide
• Add G-stain

Question 12

What chromsomal abnormality can be detected with a g stain? Explain it.

Answer 12

Philadelphia Chromosomes

• ABL gene on bottom of chromosome 9
• BCR gene on chromosome 22
• There is translocation of chromosome where ABL fuses with BCL on chromosome 22
• The product is oncogenic and it can develop into myeloid leukemia cancers.

Question 13

Describe FISH

Answer 13

FISH stands for fluorescent in situ hybridization. It also uses cultured cells and metaphase chromosomes like g -stain. However, it detects changes that are microscopic (still big but smaller than g banding). It also uses fluorescent probes to bind to DNA rather than stain.

Question 14

Method of FISH

Answer 14

• Process that mounts chromosome on slide
• Denature the DNA and probe so it is single stranded. Probe must be complementary to DNA of interest..
• Add probe. We can then see the probe DNA because it is labelled with fluorescent dye
• DNA and probe reanneal
• Observe the DNA

Question 15

What chromosomal abnormalities can FISH detect?

Answer 15

FISH tests are good for identifying microdeletions e.g. 22q11del syndrome. Prader-Willi syndrome (chromosome 15), Cri-du-chat (chromosome 5).

Usually these diseases are proved by the absense of a fluorescent marker on one chromosome as the primer cannot bind to corresponding DNA sequence due to microdeletion.

Question 16

What is the limitation of FISH, QF-PCR?

Answer 16

The cause of a disease needs to be known in order to be able to test for it by standard FISH. This is because you have to be able to design a specific probe that will bind to the DNA and show up in the region that is deleted.

Question 17

Describe QF-PCR

Answer 17

QF-PCR stands for quantitative fluorescence PCR. It uses macrosatellites and is a quick way to determine trisomies e.g. 13(patau), 18 (edwards), 21 (downs). It is therefore used to tell us how many chromosomes the patient has.

Question 18

What are microsatellites?

Answer 18

Microsatellites are repeated a lot in the genome, most of the genome is not used to make proteins and not involved in disease status.

• These repeats can be di (CACACA) , tri (CATCATCAT( or tetra, penta, hexa etc.
• They will have a variable number of repeats, we all have every single microsatellite BUT the actual number of repeats that we individually have will vary between individuals.
• These repeats are usually heterozygous
• Microsatellites always have unique flaking sequences and these allow us to recognise the microsatellite on any particular chromosome

Question 19

How does trisomy affect microsatellite?

Answer 19

With a trisomy we will see THREE copies of that microsatellite (instead of 2), in this case on three different chromosomes.

Question 20

Method of QF-PCR

Answer 20

• Tag the microsatellite using fluorescent tags

- We can then determine how many peaks which indicate how many copies of microsatellite there are

Question 21

Describe Array-CGH

Answer 21

Array-CGH is Array Comparative Genomic Hybridisation.
It looks for sub-microscopic chromosome abnormalities such as microdeletions, microduplications, CNV (copy number variations). It is mainly used to identify thigns such as developmental problems or dysmorphia as this tends to be where CNVs are responsible.

Question 22

What is an array?

Answer 22

An array is an ordered assembly of nucleic acids (probes) immobilised on a solid support e.g. Glass/ something similar to a microscope slide. Each probe is specific of a specific part of a genome.

Question 23

What are CNV (copy number variation)s?

Answer 23

CNVs are a form of microdeletion, they are talking about an increase/decrease in the number of bases.
CNVs can be protective e.g. HIV or detrimental e.g. autism. Many do nothing and are present in about 12% of the genome.
The simplest type of copy number variation is the presence or absence of a gene. An individuals genome could therefore contain two, one or zero copies

Question 24

Method Array CGH

Answer 24

• The patient and control DNA are labelled with different fluorescent dyes and applied to a microassay.
• Patient and control DNA compete to attach/anneal to the probes on the microarray slide.
• There should be equal binding to the slide probes, there should be two copies of each DNA in a normal person because the control DNA and normal person DNA is diploid. So that the relative fluorescence comes out as being equal. If there is deviation and the fluorescence of the microassay is different, then it indicates that there has been a duplication or deletion causing this change in fluorescence.
  An microarray scanner will measure the fluorescent signals.

Question 25

Describe Sanger sequencing

Answer 25

Sanger sequencing we are typically trying to target an individual gene, trying to sequence the exons.

Question 26

Sanger sequencing ingredients

Answer 26

• We need patient DNA (DNA template)
• We need DNA primers
• We need DNA polymerase (Taq)
• dNTPs (nucleotides)
  ddNTPs (has hydroxyl group, therefore extra oxygen, removed)

Question 27

Why is sanger sequencing also called dideoxy/chain termination method?

Answer 27

It needs ddNTP. The ddNTP is important because it lacks the –OH it means it cannot form a phosphodiester bond, so it stops new nucleotides being added.

Question 28

Method of Sanger sequencing

Answer 28

First, we have to isolate the DNA and amplify the genomic bit of interest (by PCR). The DNA should be free of protein and cellular debris.
The sequencing reaction:
1. Strand separation
2. Anneal primer to single strand at 3’ region (DNA polymerase acts from 5’ to 3’)
3. Extension step where dNTPs and ddNTs are added. ddNTPs are fluorescently tagged.
4. As we use Taq polymerase, complementary strand is built up using the dNTPs and randomly a ddNTP will be added which will terminate the strand and no more nucleotides can be added.

• So we end up with fragments of every possible length of our exon that we are sequencing,

The fragments are then loaded onto a gel and an electrical current seperates the fragment by weight (smaller ones move through gel faster than larger ones). At the bottom, a laser makes the fluorescent tag excited and this is detected and the sequence is built up.
The genome is then compared to a reference sequence of normal DNA.

Question 29

Describe next generation sequencing

Answer 29

• Is targeted
• Allows to sequence the whole exome or genome, rather than going in with a gene already in mind (like Sanger sequencing). You can essentially go in hypothesis free.
• Massively parallel
• Fast - ‘sequencing by synthesis’
• Generates massive amounts of data