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Repro 313 > Methods in reproduction Lecture One > Flashcards

Methods in reproduction Lecture One Flashcards

1
Q

What are two methods of amplifying DNA?

A

1) PCR

2) Cloning

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2
Q

What does PCR achieve?

A
  • Selectively Amplifying DNA to be Analysed
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3
Q

What is the rate of DNA amplification in PCR?

A

Logarithmic - so doubles ever cycle

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4
Q

How does PCR amplify DNA?

A

In vitro amplification using enzymatic processes.

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5
Q

What does the latest generation of PCR involve?

A
  • Computerised thermal cylinders, which automatically heat and cool during a cycle
  • thermostable Taq polymerase isolated from Thermus aquatics
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6
Q

What are the steps of PCR?

A

1) Denaturation
2) Annealing
3) DNA synthesis

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7
Q

What happens in denaturation?

A

The system is heated to 90-95 degrees celsius and the two strands of DNA seperate

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8
Q

What happens in the annealing stage of PCR?

A

A primer and reverse primer attach to the single strand of DNA and form brackets around the specific sequence

Around 50-65 degrees

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9
Q

What happens in the DNA synthesis stage of PCR?

A

Tay Polymerase attaches and produces a complimentary strand of DNA (72 degrees)

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10
Q

Are the temperatures consistent for all PCR?

A

No- it depends on the enzyme being used

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11
Q

Describe PCR in simple terms:

A

PCR is the process of selectively amplifying DNA by denaturation (separation of DNA strands), annealing primers and using Taq Polymerase to produce the target complimentary sequence.(DNA amplification)

This is a logarithmic reaction in a thermal cylinder.

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12
Q

What are the applications of PCR?

A
  • Substitute for cloning
  • To amplify specific regions of DNA from small amounts of material
  • Selectively detect foreign DNA sequences in tissue being tested i.e viruses
  • Analysis of highly degraded DNA
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13
Q

What are the ingredients for PCR?

A
  • Target DNA
  • Specific primers
  • Deoxyribosenucleoside triphosphate (bases)
  • Taq DNA polymerase
  • PCR buffer

NO DNA LIGASE

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14
Q

What are some characteristics of a good primer?

A
  • Must be specific
  • 18-30 base pairs
  • melting temp (56-75 C)
  • GC content 40-60%
  • 3’ end, ending with G or C
  • Avoid secondary structures
  • Avoid runs of bases
  • Avoid intraprimer homology (primers adhering to one another)
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15
Q

How can the products of PCR be prepared for analyses?

A

Gel electrophoresis

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16
Q

How does gel electrophoresis work?

A

It separates DNA fragments according to size and conformation

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17
Q

What are the types of gels and their uses in gel electrophoresis?

A

Polyacrylamide gel - single strand DNA less than 500bp
Agarose gel - 300-20,000 bp
Pulsed-Field gel - Long DNA molecules

18
Q

Whats special about agarose gel?

A

DNA moves from negative to positive terminals when a current is applied.

19
Q

How is a agarose gel read?

A

The separated DNA is stained with ethidium bromide and exposed to UV, becoming visible and reference well indicates the various mass of DNA (as short fragments move faster and therefore further within the allocated time.

20
Q

What would you do if you had non-specific product?

A

Increase the annealing temperature

21
Q

What would you do if you had no product?

A

Decrease the annealing temperature

22
Q

When would you alter the magnesium concentration?

A

When no product was present (post-lab find out what this is)

23
Q

When would you alter the amounts of primer?

A

When there is a concentration imbalance between the two?

24
Q

When would you increase the number of cycles?

A

When a small faint product is produced

25
Q

When would you decrease the number of cycles?

A

When a large indistinguishable product is produced.

26
Q

What does DNA sequencing achieve?

A

Allows you to read the nucleotides of the DNA strand

27
Q

Whats a method of DNA sequencing?

A

Replicate a target sequence using normal deoxyribosenucleoside triphosphate and a trace amount of dideoxyribosenucleoside triphosphate (these terminate the sequence) so on gel electrophoresis the fragments are separated on size and there are four troughs, one for each base thats tagged i.e A,G,C,T. So the first nucleotide in the sequence will move the furthest and increasing numbers of base pairs will be behind.

28
Q

Whats another method of DNA analysis?

A

automated sequencing using fluorescent primers. Then using a laser to identify the order of bases.

29
Q

What is NGS?

A

Next generation sequencing

30
Q

Whats an example of NGS and why is it good?

A

Massively parallel DNA sequencing.

  • High throughput
  • Reduced cost
  • Downside is hat massive amounts of sequencing data is produced.
31
Q

Whats an application of DNA sequencing?

A

Non-invasive prenatal testing using fetal DNA in the maternal plasma

32
Q

How is non-invasive prenatal testing achieved?

A
  • Fetal DNA represents 15% of the DNA in maternal plasma.
  • Uses massively parallel sequencing of maternal plasma and can detect some detail chromosome aneuploidies (abnormal chromosome number)
  • Only those at high risk in NZ are eligible to be screened.
33
Q

How is NGS used in the lab?

A
  • IN UoA they look to see if ageing oocytes DNA mutates

- Mapping genes to determine what causes disease.

34
Q

What are some examples of mRNA analysis techniques?

A 
Northern blot
in situ hybridisation
Reverse transcriptase polymerase chain reaction
Real time RT-PCR
Microarray analysis of gene expression
35
Q

Why is RT-PCR used?

A

Highly sensitive and rapid

36
Q

What is real time RT-PCR?

A

The use of a Taq man probe. This primer is designed to break become dislodged and break, becoming fluorescent when the RT reaches it during the DNA replication. The quantity of fluorescence is proportional to the amount of gene present.

37
Q

What is hybridisation?

A

Formation of double strand DNA between two single strands that are complimentary and one is labelled.

38
Q

What are some examples of hybridisation methods?

A 
Dot Blot
Southern blot
Fluorescence In Situ Hybridisation (FISH)
SNP arrays
Microarray
Array CGH
39
Q

What does microarray analysis of gene expression have in terms of characteristics?

A
  • Enables simultaneous analysis of thousands of genes

- detection of distinct gene pattern expression.

40
Q

Do the last five slides of lecture one post lab

A

do it now

Repro 313 (20 decks)

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