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Molecular 3rd year > Lecture 4: PCR > Flashcards

Lecture 4: PCR Flashcards

1
Q

what are two key applications of PCR

A
  • medical diagnosis
  • forensic investigations
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2
Q

what does PCR stand for

A

polymerase chain reaction

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3
Q

why is PCR useful

A

allows for the analysis of tiny amounts of genetic material

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4
Q

true/false gene cloning and DNA sequencing require large quantities of high-quality, intact DNA

A

true

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5
Q

what are the components needed for PCR

A
  1. template DNA
  2. DNA polymerase
  3. the four deoxynucleoside trisphosphates (dNTPs): ATGC
  4. two primers
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6
Q

what are the three steps of PCR and what happens

A
  • denaturation solution containing the DNA is heated to separate into 2 strands
  • annealing solution is cooled and primers anneal to their complementary sequences
  • extension DNA polymerase synthesizes new DNA strands by going from 5’ to 3’
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7
Q

What is typically the size for a PCR template region?

A

400-500 base pairs

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8
Q

True/false The amplified region is often longer than the template duplex

A
  • false
  • the other way around
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9
Q

How long are PCR Primers

A

20-30 nucleotides in length

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10
Q

What part of the primers are always oriented to each other

A

the 3’ end of the primer

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11
Q

how much of the primers are added to the mix

A

The primers are added to the reaction mixture in great excess to ensure pairing with any complementary sequence

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12
Q

True/ False Theoretically there is an upper limit to PCR amplification

A
  • True
  • because there is no dlifing clamp the polymerase will fall off eventually
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13
Q

How do you claculate the number of DNA after a certain number of cycles?

A

of starting DNA molecules x 2^n

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14
Q

what is the melting temp (tm)

A

the temperature at which half the helical structure is lost

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15
Q

what should Tm for the 2 primers be within of each other

A

5 degree celsius

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16
Q

what can Tm also be referred to as

A

Ta (annealing temp)

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17
Q

what is the equation for Tm

A

Tm = [4(G or C) + 2(T or A)] - 5

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18
Q

true/false the 3’ end match is most important for synthesis

A

true

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19
Q

true/false the 3’ end can have extra bases

A
  • false
  • the 5’ end can have extra
  • for example, adding restriction enzyme sites to the primer
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20
Q

what are the 2 types of primerss?

A
  • forward
  • reverse
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21
Q

where does the forward primer anneal to

A

the strand going 3’ to 5’

22
Q

where does the reverse primer anneal to

A

the strand going 5’ to 3’

23
Q

what has to be done to get the reverse primer

A

take the reerse compliment

24
Q

what are 3 key factors in PCR popularity

A

1) Availability of DNA polymerase that are resistant to high temperatures
2) Automation of temperature control heat blocks
3) Drastic decrease in the costs of thermocyclers and heat tolerant DNA polymerases

25
Q

how did PCR work in the early days

A
  • there were 3 heat blocks or water baths of diff temps
  • the tubes were manually transfered for each step
  • also new polymerases had to be added cause the e.coli polymerase was destroyed by the high temp during each denaturation step
26
Q

what polymerase is now commonly used for the process of PCr

A

Taq Polymerase

27
Q

what is the key difference between e.coli polymerases and Taq polymerase

A
  • e.coli polymerase is destroyed by high temp in the denaturation
  • Taq polymerase comes from a bacterium called thermus aquaticus, which is found in hot springs, so it can tolerate the high temps
28
Q

what machine is PCR performed in

A

thermocycler

29
Q

what is a thermocycler

A

an electronically controled heat block that is programmed to change automatically to the diff temps needed for each PCR cycle step also means that you don’t have to be physically present for every single step

30
Q

what happens in gel electrophoresis

A
  • dna samples are inserted into wells on the edge of the gel
  • using an electrical current, the DNA fragments move towards the positive pole according ot their size
31
Q

________ (smaller/ larger) fragments move faster in gel electrophoresis

A

smaller

32
Q

________ (smaller/ larger) fragments move slower in gel electrophoresis

A

larger

33
Q

what does a band represent in gel electrophoresis

A

represents the DNA of a particular size

34
Q

true/false the size and amount of DNA is correlated

A
  • false
  • just because you have a 1000 bp fragment doesn’t mean you have more than what is in a 400 bp fragment
35
Q

what represents the amount of DNA in gel electrophoresis

A

amount of dna is represented by the brightness of the band

36
Q

what is used to make the gel in gel electrophoresis

A
  • agarose powder
  • TAE buffer
  • indicator
37
Q

true/false In gel electrophoresis size relates to distance traveled

A

true

38
Q

what is used in gels to determine the size od the band

A

Premade ladders with bands of known sizes

39
Q

what is what is ethidium bromide

A

an intercalating agent (ie inserts itself between DNA bases)

40
Q

true/false bands cannot be separated and recovered for later use

A
  • false
  • they can!
41
Q

what does RT PCR stand for

A

reverse transcriptase polymerase chain reaction

42
Q

what is the purpose of RT PCR

A

to isolate total RNA from a sample, remove gDNA, use polymerase that uses an RNA template to make a DNA strand (cDNA)

43
Q

what does qPCR stand for

A

quantitative polymerase chain reaction

44
Q

what is the goal of qPCR

A

measured the amplification in real time during amplification

45
Q

true/false enzymes exist that can form RNA directly from RNA templates

A

false

46
Q

true/false PCR can be used for diagnostic purposes but not forensic purposes

A
  • false
  • can be used for both
47
Q

how can we use qPCR to determine the starting conc of the mRNA of interest

A

by monitoring the fluorescence

48
Q

what is sequence analysis

A

the comparison and alignment of sequences with control and database sequences

49
Q

what is used for DNA profiling

A

short tandem repeats

50
Q

what happens when you increase the number of regions investigated

A
  • you increase the variability,
  • but you end up decreasing the probability of finding another with the same combination of repeats
51
Q

the length of the stretches in DNA of STR vary depending on what

A

depending on the number of copies of the repeat found at a specific reegion (locus) of DNA

52
Q
A

Molecular 3rd year (9 decks)

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