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Yr 2 - EMS > Genome Analysis Methods > Flashcards

Genome Analysis Methods Flashcards

1
Q

What is PCR?

A

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

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2
Q

How does PCR work?

A

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

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3
Q

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

A

Designing small synthetic primers –> oligonucleotides

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4
Q

How is DNA synthesised in PCR?

A

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

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5
Q

What is the first stage of PCR?

A
  1. Heat denaturation
  • Temperature increased to 94 degrees
  • This denatures DNA, causing hydrogen bonds to break, leaving 2 single stranded DNA molecuels
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6
Q

What is the second stage of PCR?

A
  1. Primer annealing

Temperature lowered to annealing temperature of 55 degrees

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

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7
Q

What is the third stage of PCR?

A

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
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8
Q

What occurs after primer extension?

A

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

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9
Q

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

A

It doubles –> the process is exponential

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10
Q

What is PCR used to detect?

A

Presence of specific sequences:

  • Genomic sequence
  • Virus
  • Calculating foetal or tumour DNA
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11
Q

What is Sanger sequencing?

A

The sequencing technology used in the human genome mapping project

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12
Q

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

A

PCR products

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13
Q

What ingredients are needed in Sanger sequencing reaction?

A
  • 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)
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14
Q

What is purpose of ddNTPs?

A

Chemically modified so they cannot be extended and therefore terminate reaction

Labelled with fluorescent dyes

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15
Q

What does Sanger sequencing result in?

A

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

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16
Q

What is Next Generation Sequencing (NGS)?

A

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.

17
Q

What is whole exome sequencing?

A

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
18
Q

What is purpose of whole exome sequencing?

A

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

19
Q

What is meant by large scale genome pathology?

A

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

  • Chromosome number
  • Chromosome structure
20
Q

What is karyotyping?

A

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.

21
Q

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

A

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

22
Q

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

A

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

23
Q

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

A
  1. G-banding
  2. NGS
  3. Microarrays (aCGH)
24
Q

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

A
  1. FISH
  2. MLPA
  3. QF-PCR
25
Q

When would targeted testing be used?

A

When the causative gene variant has usually already been identified

26
Q

What is whole genome sequencing (WGS)?

A

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 variants associated with the disease, which can be found using other methods such as whole genome sequencing.

27
Q

What is disadvantage of WGS?

A

Generates many variants - analysis is very tricky

28
Q

Why is PCR needed for DNA anaylsis?

A

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.

29
Q

Which anaylsis technique is used in DNA fingerprinting?

A

PCR

30
Q

Which analysis method is used in the detection of HIV?

A

PCR

31
Q

What is the function of DNA sequencing?

A
  • 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.
32
Q

What are dNTPs?

A

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

33
Q

What are ddNTPs?

A

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

34
Q

What is the role of ddNTPs in sequencing?

A

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.

35
Q

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

A

ddNTPs

36
Q

What occurs during metaphase?

A

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.

37
Q

What is copy number variation?

A

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

Yr 2 - EMS (60 decks)

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