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Genetics > Lecture 5 - Molecular Diagnostics > Flashcards

Lecture 5 - Molecular Diagnostics Flashcards

1
Q

What are molecular diagnostics?

A

Various molecular biology techniques used to analyze biological markers in the genome and proteome

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2
Q

What are molecular diagnostics used for?

A
  • Monitoring disease
  • Detecting disease risk
  • Identify therapies
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3
Q

Pharmacogenomics

A

Using molecular biology technologies to predict a response to drugs (differences in enzymes involved in metabolism)

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4
Q

How are molecular diagnostics used in infectious disease?

A
  • Identify infection status
  • Identify strains
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5
Q

How are molecular diagnostics used in cancer?

A
  • Breast cancer risk (BRCA1/2)
  • Diagnostics
  • Oncogene mutations
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6
Q

Which factors are leading to the growth of molecular diagnostic techniques?

A
  • Zoonotic diseases
  • Animal agriculture
  • Pet adoption and healthcare
  • Pet insurance
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7
Q

Molecular diagnostic techniques for DNA/RNA

A
  • Sanger sequencing
  • SNP microarrays
  • Next-generation sequencing
  • PCR
  • Isothermal amplification
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8
Q

Molecular diagnostic techniques for proteins

A
  • ELISA (enzyme-linked immunosorbent assay)
  • Proteomics
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9
Q

PCR is used to

A
  • Amplify target DNA and RNA
  • Test for the presence or quantity of DNA/RNA in a sample
  • Targeted genotyping (single SNPs or insertion/mutation)
  • Facilitate other applications such as sanger sequencing or DNA metabarcoding (by amplifying DNA)
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10
Q

What is needed for PCR?

A
  • A DNA template
  • A pair of synthetic single stranded DNA primers
  • dNTPs (deoxyribonucleotide triphosphates)
  • A heat stable polymerase (Taq)
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11
Q

Taq polymerase

A

A heat-stable polymerase from the bacteria Thermus aquaticus

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12
Q

Downsides of Taq polymerase

A

Unable to repair base pair errors during replication (cannot proofread)

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13
Q

The amount of DNA ____ every cycle of PCR

A

Doubles

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14
Q

How can molecular diagnostics determine disease risk?

A

Looks at the presence/absence of SNPs that cause disease

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15
Q

What is the role of the heat-stable polymerase in PCR?

A

To extend the primer

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16
Q

Binding requirements for PCR primer design

A

Must only bind at one location on the genome

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17
Q

Length requirements for PCR primer

A

18-25 nucleotides, helps avoid having a similar sequence elsewhere that the primer could bind to

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18
Q

Melting temperature requirements for PCR primer design

A

The temperature at which the pair of primers dissociate from the template should be within +/-5 degrees celcius

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19
Q

Primer dimers

A

When forward and backward primers bind together - must be avoided since the primer won’t work if it can bind with itself

20
Q

Structure requirements for PCR primer design

A

Structure must be avoided

(idk what this means tbh but its in the slides so if anyone else knows let me know - Aimee)

21
Q

What allows the visualization of PCR results

A

Gel electrophoresis

22
Q

How does gel electrophoresis work?

A
  • Separates DNA molecules by length (smaller molecules travel faster)
  • DNA is loaded onto agarose gel and will result in bands at different locations which indicates where different nucleotides are located in the strand
23
Q

Why is a negative control used in PCR

A

Contamination is an issue in PCR, a negative control ensures that the sample was not contaminated

24
Q

What is capillary electrophoresis?

A

Capillary electrophoresis involves separating segments based on size, similar to gel electrophoresis. This can occur in the same machine as sequencing (ex. Sanger sequencing).

25
Q

A limitation of PCR is that it requires prior knowledge of ____.

A

Some of the target sequence

26
Q

List the limitations of PCR

A
  • Requires some knowledge of the sequence
  • Contamination
  • Taq polymerase does not have the ability to proofread
  • Some samples contain PCR inhibitors that can inhibit the reaction
  • Longer segments cannot be reliably amplified
27
Q

Up to what length can PCR reliably amplify?

A

2000bp

28
Q

What are some PCR inhibitors that can be present within samples?

A
  • Humic acid
  • Urea
  • Calcium ions
29
Q

Which veterinary infections can PCR detect?

A
  • Avian Influenza A
  • Rift Valley Fever Porcine Viruses
  • Bovine Diarrhea Virus
  • Sheep & Goat Poxvirus
  • African Horse Sickness
30
Q

What is the abbreviated name for reverse transcription PCR

A

RT-PCR

31
Q

What is the abbreviated name for real time PCR

A

qPCR aka. quantitative PCR

32
Q

What is RT-PCR?

A

RT-PCR uses the enzyme reverse transcriptase to create a DNA template from a strand of RNA that can then be amplified.

33
Q

What is RT-PCR used for?

A

RNA viruses or gene transcription.

34
Q

Transcriptomics

A

A technique to look at all of the RNA in a sample. It uses the poly-A tail that is added onto RNA during transcription to amplify all of the RNA in a sample by converting it to a DNA complement.

35
Q

What method is used to look for transcription of a specific gene?

A

RT-PCR, a specific primer is used to look for a target sequence.

36
Q

What method is used to look for all RNA transcription in a sample?

A

RNA-seq (transcriptomics)

37
Q

What is qPCR?

A
  • Quantifies the amount of DNA in a sample.
  • Uses fluorescence to determine the amount of DNA compared to a known sample.
38
Q

What are the benefits of qPCR?

A

Faster, more sensitive, more accurate quantitation than PCR.

39
Q

What is DNA metabarcoding?

A

It is a process that can determine which species are present in a sample with mixed DNA.

40
Q

What are the steps for DNA metabarcoding?

A
  1. DNA extraction
  2. PCR amplification
  3. Next generation sequencing of PCR products
  4. Compare data from sequencing to a database with reference sequences
  5. Infer species composition
41
Q

What is required for DNA metabarcoding to work?

A

The sequence of the species present in the sample must be in the database.

42
Q

How is isothermal amplification different from normal PCR?

A

Isothermal amplification can occur at a constant temperature whereas PCR requires temperature cycling.

43
Q

Isothermal amplification

A

Loop-mediated isothermal amplification is a single-tube technique for the amplification of DNA.

44
Q

What technology was used in the Covid rapid test kits?

A

LAMP (isothermal amplification)

45
Q

How does loop-mediated isothermal amplification (LAMP) work?

A
  • 4 to 6 primers are used.
  • DNA amplification occurs due to elongation of the primer which separates the target strand.
  • A self-hybridizing loop forms on the new strand due to the inclusion of a reverse complimentary sequence.
  • The process repeats and a loop forms on the opposite side, forming a dumbbell structure.
  • The products can be detected in a variety of ways.
46
Q
A

Genetics (10 decks)

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