Genome Analysis Methods

Question 1

What is PCR?

Answer 1

Polymerase Chain Reaction: A method widely used to rapidly make millions to billions of copies of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail

Question 2

How does PCR work?

Answer 2

‘In vitro’ synthesis of large amounts of DNA by copying from small starting quantities

Question 3

How is the region you want to amplify defined in PCR?

Answer 3

Designing small synthetic primers –> oligonucleotides

Question 4

How is DNA synthesised in PCR?

Answer 4

By a DNA ‘polymerase’ enzyme from single ‘monomers’ (deoxy-ribonucleotides)

Question 5

What is the first stage of PCR?

Answer 5

1. Heat denaturation

• Temperature increased to 94 degrees
• This denatures DNA, causing hydrogen bonds to break, leaving 2 single stranded DNA molecuels

Question 6

What is the second stage of PCR?

Answer 6

2. Primer annealing

Temperature lowered to annealing temperature of 55 degrees

Primers can then anneal to single stranded templates (work in 3’ to 5’ direction)

Question 7

What is the third stage of PCR?

Answer 7

Primer extension

• Temperature of 72 degrees (thermostable DNA polymerase ‘Taq polymerase’ is used)
• DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand in the 5’ to 3’ direction

Question 8

What occurs after primer extension?

Answer 8

Process is started again –> heat denaturation at 94 degrees, then primer annealing and extension

This time the primers have attached to the newly synthesised DNA as well as the original templates

Question 9

What happens to the number of templates with every cycle of PCR?

Answer 9

It doubles –> the process is exponential

Question 10

What is PCR used to detect?

Answer 10

Presence of specific sequences:

• Genomic sequence
• Virus
• Calculating foetal or tumour DNA

Question 11

What is Sanger sequencing?

Answer 11

The sequencing technology used in the human genome mapping project

Question 12

What is often used as template DNA in a Sanger sequencing reaction?

Answer 12

PCR products

Question 13

What ingredients are needed in Sanger sequencing reaction?

Answer 13

• Single stranded DNA template
• 1 DNA primer; can be same one used in PCR reaction or any primer that anneals to template
• DNA polymerase
• Nucleotides (dNTPs)
• Dye terminator nucleotides (ddNTPs)

Question 14

What is purpose of ddNTPs?

Answer 14

Chemically modified so they cannot be extended and therefore terminate reaction

Labelled with fluorescent dyes

Question 15

What does Sanger sequencing result in?

Answer 15

Results in the formation of extension products of various lengths terminated with dideoxynucleotides at the 3′ end. The extension products are then separated by Capillary Electrophoresis or CE.

Results in colourful electropherogram which shows sequence of DNA

Question 16

What is Next Generation Sequencing (NGS)?

Answer 16

Term that describes a DNA sequencing technology which has revolutionised genomic research. Using NGS an entire human genome can be sequenced within a single day.

Question 17

What is whole exome sequencing?

Answer 17

Together, all the exons in a genome are known as the exome, and the method of sequencing them is known as whole exome sequencing.

1. DNA is transcribed into RNA
2. All the introns are removed –> left with coding sequence only (exons)
3. Sequence the exonic DNA

Question 18

What is purpose of whole exome sequencing?

Answer 18

To identify genetic variants that alter protein sequences, and to do this at a much lower cost than whole-genome sequencing.

Question 19

What is meant by large scale genome pathology?

Answer 19

At the level of the chromosome –> reaarangements of the genome

• Chromosome number
• Chromosome structure

Question 20

What is karyotyping?

Answer 20

Analysis of the entire chromosome complement through the microscope. Dividing cells are harvested during metaphase, the time of greatest chromosome condensation, and chromosomes are visualised by staining to elicit banding patters.

Question 21

What is comparative genome hybridisation (Array CGH)? What is it used for?

Answer 21

A molecular cytogenetic method for analysing copy number variations (e.g. deletions) in the DNA of a test sample compared to a reference sample, without the need for culturing cells

Question 22

Will comparative genome hybridisation work in a balanced translocation? Why?

Answer 22

No - still same amount of DNA but just in the wrong place

Question 23

What methods are used to detect copy number variation at the level of the whole genome?

Answer 23

1. G-banding
2. NGS
3. Microarrays (aCGH)

Question 24

What methods are used to detect copy number variation at the level of targeted testing?

Answer 24

1. FISH
2. MLPA
3. QF-PCR

Question 25

When would targeted testing be used?

Answer 25

When the causative gene variant has usually already been identified

Question 26

What is whole genome sequencing (WGS)?

Answer 26

Exome sequencing is only able to identify those variants found in the coding region of genes which affect protein function. It is not able to identify the **structural and non-coding variant**s associated with the disease, which can be found using other methods such as **whole genome sequencing.**

Question 27

What is disadvantage of WGS?

Answer 27

Generates many variants - analysis is very tricky

Question 28

Why is PCR needed for DNA anaylsis?

Answer 28

Because significant amounts of a sample of DNA are necessary for molecular and genetic analyses, studies of isolated pieces of DNA are nearly impossible without PCR amplification.

Question 29

Which anaylsis technique is used in DNA fingerprinting?

Answer 29

PCR

Question 30

Which analysis method is used in the detection of HIV?

Answer 30

PCR

Question 31

What is the function of DNA sequencing?

Answer 31

* Determining the **order of the four bases** that make up the DNA molecule * The sequence tells you the kind of genetic information that is carried in a particular DNA segment. * E.g. scientists can use sequence information to determine which stretches of DNA **contain genes** and which stretches carry **regulatory instructions**, turning genes on or off. * In addition, sequence data can highlight **changes in a gene that may cause disease.**

Question 32

What are dNTPs?

Answer 32

There are four types of dNTP, with each using a different DNA base: adenine (dATP), cytosine (dCTP), guanine (dGTP), and thymine (dTTP).

Question 33

What are ddNTPs?

Answer 33

The ddNTPs are the artificially synthesised special type of nucleotides used in the Sanger sequencing technique.

Question 34

What is the role of ddNTPs in sequencing?

Answer 34

**ddNTPs mediated-chain termination method:** the signal given by the termination is recorded by the computer and generates the peak of various nucleotides as per the fluorescence emitted. The end of the ddNTP-dideoxynucleotides are labeled with the **color dye**, the color signal is recorded by the computer.

Question 35

What is involved in sequencing that is not involved in PCR?

Answer 35

ddNTPs

Question 36

What occurs during metaphase?

Answer 36

Metaphase is a stage in the cell cycle where all the **genetic material is condensing** into chromosomes. These chromosomes then become **visible**. During this stage, the **nucleus disappears** and the **chromosomes appear in the cytoplasm** of the cell.

Question 37

What is copy number variation?

Answer 37

A copy number variation (CNV) is when the number of copies of a particular gene varies from one individual to the next.