PRIS > DNA diagnostics > Flashcards

DNA diagnostics Flashcards

(39 cards)

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1
Q

Why do we use DNA for diagnostics?

A
  • DNA is relatively stable
  • DNA can be replicated in the lab (PCR) allowing for high sensitivity/ low initial sample needed
  • DNA change is critical in genetic disorders, cancer
  • most pathogens have DNA for genome
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2
Q

How do we get DNA for some DNA tests?

A
  • no DNA purification is needed
  • e.g., colony PCR of bacteria
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3
Q

How would you get DNA for genetic disorder tests on dogs?

A
  • mouth swabs common (epithelial cells)
  • and less commonly blood samples
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4
Q

When testing for non-leukocyte cancers what is required?

A
  • biopsy
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5
Q

What is critical when sampling for DNA testing?

A
  • no contamination is critical
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6
Q

What would you have to do for blood sample when testing for DNA?

A
  • EDTA used as anti-coagulant as it inhibits DNases
  • would not use heparin as this inhibits Taq polymerase by binding it
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7
Q

What has become increasingly important in disease control?

A
  • environmental DNA sampling
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8
Q

What are the steps to the amplification technique for PCR?

A
  1. relies on DNA template
  2. specific primers to site of interest/specific to pathogen
  3. multiple cycles of denaturing, annealing, extension
  4. gel electrophoresis to reveal result
  5. presence/absence
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9
Q

Name two RNA viruses?

A
  • influenza
  • west Nile virus
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10
Q

For PCR with RNA viruses what is the additional step that must happen first (before the normal process)?

A
  • Need to use reverse transcriptase enzyme
  • to turn RNA > dsDNA
    = reverse transcription PCR
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11
Q

Why do some labs prefer to use Real time or Quantitative PCR?

A
  • for some studying we want more than just presence or absence
  • also faster - no gel running - reduces time and man power
  • magnitude (4x more of virus genome in this individual etc.)
  • amount of genome broadly equates to amount of viable pathogen - there are exceptions though
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12
Q

What is a type of qPCR?

A
  • SYBR green method is simplest
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13
Q

What does the SYBR green method entail?

A
  • regular PCR reaction for one specific region
  • SYBR green binds to all newly synthesised double stranded DNA
  • when bound SYBR green is fluorescent
  • qPCR machine measures fluorescent in tube
  • detects fluorescence at the end of each denature, anneal and extend cycle
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14
Q

What are the uses of qPCR?

A
  • veterinary parasitology
  • molecular biology
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15
Q

What are the limitations to qPCR?

A
  • contamination is an issue (all amplification methods will suffer with this)
  • can only measure one (SYBR green) or < 10 (taqman) transcripts per tube
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16
Q

Due to the limitations of qPCR what do we use for a greater global understanding?

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A
  • SNP microarray
  • next gen sequencing
17
Q

What are SNP arrays a type of?

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A
  • one type of DNA microarray
18
Q

What are SNP arrays heavily used in?

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A
  • heavily used in breeding programmes
19
Q

What does SNP array rely on?

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A
  • relies on knowing the SNPs already from sequencing studies
20
Q

What does a SNP array look like?

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A
  • glass slide with little squares containing dots that have a bit of DNA with a specific SNP - which can be used to work out what the animals alleles are
21
Q

What is first added onto the SNP array?

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A
  • 100,000s of short, specific single stranded DNA oligos spotted onto glass slide
  • each added at a particular grid position
22
Q

What are DNA oligos (25-50bp) synthesised to represent?

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A
  • particular SNP regions
23
Q

Once the DNA oligos have been added - What is the third part to a SNP array?

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A
  1. Take a sample. Multiples copies of the genome (DNA from multiple cells) are sheared (split apart)
  2. fluorophore (dye) added to DNA fragments
  3. Hybridisation with SNP array - denatured to single strands to they can complement their SNP
24
Q

What does a Affymetrix SNP assess?

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A
  • strong/weak hybridisation
    = strong signal (fluorescence) = correct allele
    = weak signal is due to not as full complementary = incorrect allele
25
How do illumima SNP arrays work differently to Affymetrix SNP's?
- Still DNA hybridisation but utilising different dyes for different bases at the SNP position
26
What do Illumina SNP arrays determine for each sample?
- Presence/absence for each SNP
27
In illumina SNP array what is required to ID associations?
- computational and statistical analysis
28
What are the two ways we can DNA sequence?
- sanger sequencing - next-gen sequencing
29
What are both sanger and next gen sequencing used for?
- sequencing specific regions - whole genome shotgun sequencing
30
What are the benefits to using DNA sequencing over other methods?
- sequencing will enable understanding of relationships between samples - pathogen spread - whole genome sequencing allows for novel mutations to be IDed - can be especially good for mixed samples
31
What is sanger sequencing?
- old technique - used for all the early sequencing projects - horse, chicken, dog, human
32
What does sanger sequencing do?
- incorporation of terminator bases (ddNTPs) stops new bases being added, containing fluorophores (depending on what base) during in vitro DNA replication
33
What does next-gen sequencing use?
- illumina/soleca, 454, SOLiD
34
What are the advantages if using next-gen sequencing?
- cheaper - far less DNA needed
35
What are the disadvantages for using illumina in next-gen sequencing?
- short reads - higher error rate
36
What is the future of next-gen sequencing?
- future is single molecular sequencing (PacBio, Nanopore)
37
What are the uses of DNA sequencing?
- compare individual/breeds with whole genome sequencing
38
What is the aim of DNA sequencing?
- to identify base pairs within the genome associated with milk production, embryonic death, disease
39
What can DNA sequencing reveal?
- why cancers develop - breed specifics - what genes specifically are causing cancers - what drugs/ MOA would would work best
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