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Week 1 - Medical Genetics > DNA and Chromosomal Analysis Methods > Flashcards

DNA and Chromosomal Analysis Methods Flashcards

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1
Q

What are the features of DNA?

A
  • Double stranded helical structure
  • Arranged in an anti-parallel fashion
  • Made up of nucleotides
  • wrapped around histone proteins
  • DNA coils around itself (supercoil)
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2
Q

What are the components of a nucleotide?

A

Deoxyribose sugar, phosphate group, nitrogenous base

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3
Q

What are the 4 nitrogenous bases?

A

Adenine, Guanine, Cytosine, Thymine (Uracil)

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4
Q

Example of Purines?

A

Guanine, Adenine

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5
Q

Example of Pyrimidines?

A

Cytosine, Thymine, Uracil

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6
Q

How many hydrogen bonds are formed between Adenine and Thymine?

A

2

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7
Q

How many hydrogen bonds are between Cytosine and Guanine?

A

3

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8
Q

What forms the backbone of the DNA molecule?

A

Sugar phosphate backbone (Covalent bond)

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9
Q

What happens to DNA molecule if it is heated to 95 - 100 degrees Celcius?

A

The hydrogen bonds will be broken but the covalent bond is still stable.

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10
Q

What is Chargaff’s rule?

A

The ratio of a nucleotide is the same as the one that it is paired to.

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11
Q

The human genome has around 3 000 Million base pairs, how many of them truly code for proteins?

A

Around 1.1%

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12
Q

4% of the human genome are non-protein coding genes that play a crucial role as enhancers, silencers and insulators. What are they?

A

Enhancers : enhance the activity of a gene by activating promoters
Silencers : repress the activity of a gene
Insulators : Prevent enhancers from activating other promoters - to prevent a gene from being influenced by activation or repression of its neighbouring regulators

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13
Q

What are RNAs?

A

Ribonucleic acid

  • pentose sugar has hydroxyl molecules at Carbon 2 and Carbon 3
  • Uracil is present instead of Thymine as pyrimidine
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14
Q

Types of RNA?

A

mRNA. tRNA, rRNA (form ribosomes), microRNA (regulates gene expression)

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15
Q

Types of nucleotides?

A

Deoxyribonucleotide - Hydroxyl molecule is at Carbon 3
Ribonucleotide - Hydroxyl molecules at Carbon 2 and 3
Didehydroxyribonucleotide - no hydroxyl molecules at carbon 2 and 3

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16
Q

What are the DNA-based Detection Methods?

A
  1. Detection of Point Mutation
    a) DNA sequencing
    - Massively parallel sequencing
    - Sanger method
    b) Allele-specific PCR using Allele-Specific Oligonucleotides
    - Amplification Refractory Mutation System (ARMS)
    - Oligonucleotide Ligation Assay (OLA)
    c) Restriction Enzymes + gel electrophoresis
  2. Detection of submicroscopic duplications and deletions
    a) Multiplex Ligation-dependent Probe Amplification (MLPA)
    b) Multiplex PCR
    c) Array Comparative Genomic Hybridisation (aCGH)
  3. Rapid detection of aneuploidies
    - Quantitative Fluorescent PCR
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17
Q

What is DNA sequencing?

A

The determination of the precise sequence of nucleotides

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18
Q

What is genetic amplification?

A

The process of increasing the copies of DNA from almost undetectable amount to a significant amount using enzymes and other reagents.

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19
Q

What process is used to amplify genetic material?

A

Polymerase chain reaction

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20
Q

What are the required substances for PCR?

A
  1. DNA template
  2. Thermostable DNA polymerase from thermophilic bacteria
  3. 2 Oligonucleotide primers
  4. Free deoxyribonucleotides
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21
Q

What are the steps involved in PCR?

A
  1. Denaturation
    - Heats up to 95 degrees Celcius
    - Breaks up Hydrogen bonds
  2. Annealing
    - Primers are added when the temperature is 55 degrees Celcius
    - Forward and Reverse Primers

3, Extension

  • DNA polymerase is used
  • Binds to annealed primers
  • Reads the parent strands from 3’ to 5’ (downstream)
  • the new DNA is synthesized from 5’ to 3’
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22
Q

Why does the amount of DNA amplified reach a plateau level after 2 - 3 hours?

A
  • depletion of reagents & substrates

- accumulation of inhibitory products

23
Q

What are the ways to check the amplified DNA products?

A
  1. Stain the DNA using ethidium bromide

2. Direct visualisation of DNA under UV after gel electrophoresis

24
Q

What are the methods of detecting point mutations?

A

a) DNA sequencing
- Sanger method
- Massively parallel sequencing

b) Allele-specific PCR using Allele-specific oligonucleotides
- Amplification Refractory Mutation System (ARMS)
- Oligonucleotide Ligation Assay

c) Restriction Enzymes and gel electrophoresis

25
Q

What is the main principle behind the Sanger method of DNA sequencing?

A

Termination of DNA elongation by adding dideoxyribonucleotides that do no allow addition of nucleotides after it because it does not have a hydroxyl group at the 3’ Carbon to bind with the phosphate group.

26
Q

Substances for the Sanger Method DNA sequencing?

A
  1. DNA template
  2. Thermophilic DNA polymerase
  3. Single oligonucleotide primer
  4. Deoxyribonucleotide
  5. Dideoxyribonucleotide with fluorescent label
27
Q

What is the mechanism of Sanger method?

A
  1. DNA amplification
  2. Termination of DNA elongation by dideoxyribonucleotides.
  3. Fragments of DNA are subjected to gel electrophoresis.
  4. DNA fragments are negatively charged so they move to the positive terminal (anode)
  5. The fragments are separated according to their weight
  6. Computer determines the sequence
    TO CHECK FOR MUTATIONS
  7. Forward and Reverse primers are used
  8. Generated complimentary strands are checked for mutations by comparing them with a known reference
28
Q

What are the limitations of Sanger Method of DNA sequencing?

A

Can sequence only short fragments of DNA up to 500 base pairs.

29
Q

What is principle of Massively Parallel Sequencing?

A

Labelled deoxyribonucleotides are added to the chain of elongation DNA and a picture is taken each time by the computer once of nucleotide has been incorporated into the chain. Can elongate different segments of DNA at a time.

30
Q

What is the limitation of Massively Parallel Sequencing technique?

A

The DNA that is read by the machine is shorter than that for Sanger method.

31
Q

What is the main principle behind Amplification Refractory Mutation System?

A

Specificity of DNA base pairing using different primers. One is a wild primer and another one is from a known mutant primer.

32
Q

What are the procedures of Amplification Refractory Mutation System?

A
  1. 2 test tubes are set up for complementary PCR reactions
  2. First test tube has a wild primer added to it.
  3. Second test tube has a mutant primer added to it.
  4. The 2 test tubes are left for 2 - 3 hours for amplification.
  5. Compare the test tubes.

If test tube 1 has more contents than test tube 2: Gene is homozygous for wild base pairs.
If test tube 1 and 2 have amplified contents. Gene is heterozygous with the mutant present.
If test tube 2 has amplification. Gene is homozygous mutant

33
Q

What are the steps involved in Oligonucleotide Ligation Assay?

A
  1. PCR amplification
    - add a wild primer and a known mutant primer
    - Mutant DNA strand cannot elongate further - shorter fragments
  2. Ligation reaction
    - addition of primers with fluorescent marker
    - ligation of the primers
    - Gel electrophoresis : long strands - wild type
34
Q

State one condition that can be diagnosed using Oligonucleotide Ligation Assay.

A

Cystic Fibrosis

35
Q

What are the methods used to detect submicroscopic duplications and deletions?

A
  • Multiplex Ligation-dependent Probe Amplification

- Array Comparative Genomic Hybridization

36
Q

What are the steps involved in Multiplex Ligation-dependent Probe Amplification

A
  1. A reagent containing 2 Probe Oligonucleotides are added
    - DYE+FORWARD PRIMER + PART 1 of the PROBE OLIGONUCLEOTIDE
    - PART 2 of the PROBE OLIGONUCLEOTIDE + stuffer genes + reverse primer
  2. Denaturation of DNA
  3. Probes bind to targeted sites
  4. Ligation of Part 1 and Part 2 of the oligonucleotide probes
  5. Gaps in between the two probes determine the size of the fragments
  6. Amplification of DNA (including the probe pairs)
  7. Analysis of amplified DNA - The fragments contain different lengths because the gaps between the 2 probes are different
37
Q

What are submicroscopic changes?

A

Changes to the DNA that are smaller than 4 000 000 base pairs and cannot be seen using light microscopy

38
Q

What are the limitations of Multiplex Ligation-Dependent Probe Amplification?

A
  1. Cannot detect areas that are unbound by the probes

2. Normal variations will affect the results

39
Q

What are the procedures of Array Comparative Genomic Hybridisation?

A
  1. Patient and Control DNA are labelled with different fluorescent dyes and applied to microarray
  2. Patient and Control DNA compete to attach to the microarray.
  3. The microarray scanner measures fluorescent signals.
  4. Computer analyses the data.
40
Q

What are the limitations of Array Comparative Genomic Hybridization?

A
  1. Cannot detect mosaicism

2. Normal variation affects results

41
Q

What is the method used to detect aneuploides?

A

Quantitative Fluorescent PCR

42
Q

What is an aneuploid?

A

Abnormal number of chromosomes that is not a multiple of 23.

43
Q

State some examples of aneuplod.

A
  1. Trisomy 13 : Patau Syndrome
  2. Trisomy 18 : Edward Syndrome
  3. Trisomy 21 : Down Syndrome
44
Q

What is the mechanism behind quantitative fluorescent PCR?

A
  1. Several short tandem repeats over the chromosomes are amplified by PCR using primer labelled with fluorescent dyes.
  2. Automated sequencer detect size ad abundance of DNA
  3. Computer plots out graphs
    - peaks : dosage
    - number of peaks : number of chromosomes
45
Q

What are the methods used to detect Chromosomal Abnormalities?

A
  1. Karyotyping

2. Fluorescence In Situ Hybridization - detect smaller specific deletions up to 1 - 2 Million base pairs

46
Q

What specimens are required for Cytogenetics?

A

White blood cells

47
Q

What are G bandings?

A

The different densities of chromosomes that are visible after using Giemsa stain.

48
Q

What are EDTA tubes for?

A

DNA test

49
Q

What are Heparin Tubes for?

A

Cytogenetic test

50
Q

What is a karyotype?

A

A complete set of chromosomes in a specific individual that is arranged according to size

51
Q

What are the limitations of karyotyping?

A
  • small subtle chromosomal abnormalities cannot be detected (relies on G Banding)
  • normal variations affect G banding patterns
52
Q

What are the limitations of Fluorescence In Situ Hybridization?

A
  • small deletions are undetected

- regions larger than the probe escapes detection

53
Q

What are euchromosome and heterochromosome?

A

Euchromosome - active chromosome

Heterochromosome - inactive chromosome

Week 1 - Medical Genetics (5 decks)

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