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Principles Of molecular Biogy > 3. PCR Reaction > Flashcards

3. PCR Reaction Flashcards

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1
Q

Apart from identical twins, all orgnaisms are

A

Genetically unique

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2
Q

Among species, what sets us apart is

A

The possession of unique genes

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3
Q

In order to identify species we must be able to

A

Detect their unique genes

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4
Q

The problem with identifying unique genes is that unique genes are not visible unless

A

We make millions of copies of it using PCR

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5
Q

PCR

A

A technique used to amplify a specific region of DNA
It involves repeated cycles of denaturation, annealing and elongation using DNA Polymerase enzymes so that would result in exponential amplification of target DNA sequence

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6
Q

One successfully visualized, the copies are detected by

A

Gel electrophoresis

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7
Q

DNA polymerase is an enzyme that

A

Duplicates DNA
Before a cell divides, its Dna must be duplicated

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8
Q

PCR being a chain reaction means that

A

The product of a reaction is used to amplify the same reaction

Results in rapid increase in the product

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9
Q

Polymerase Chain Reaction (PCR) uses principles of DNA replication to exponentially copy or amplify

A

genes or DNA segments

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10
Q

Sequence to be copied is called

A

the target DNA/ sequence

(viral RNA can also be used)

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11
Q

what can be used to be copied in PCR reactions

A

DNA sequence
or viral RNA

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12
Q

amplicon

A

The amplified fragment

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13
Q

PCR Requirements

A
  1. template DNA
  2. dNTPs
  3. Primers
  4. polymerase enzyme
  5. pcr reaction buffer
  6. MgCL2
  7. Thermal cycler/PCR machine
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14
Q

the target DNA is

A

the sequence to be copied

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15
Q

The target DNA usually originates from

A

the cells of the organsim

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16
Q

how do you get template DNA for PCR

A

The target DNA usually originates from the cells of an organism

Genomic DNA is extracted from the organism which contains the target sequence

whole cells can also be used

Strands must be separated ie single stranded for amplification to occur

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17
Q

for amplification to occur DNA strands must be

A

single stranded

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18
Q

dNTPs are the

A

raw materials necessary to synthesize DNA

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19
Q

how many dNTPs are there

A

there are 4 dNTPs for each base in DNA

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20
Q

what are the 4 dNTPs

A
  1. deoxyadenosine 5-triphosphate (dATP)
  2. Deoxycytidine 5’-triphosphate (dCTP)
  3. Deoxyguanosine 5’triphopshate (dGTP)
  4. Deoxythymidine 5’ triphosphate (dTTP)
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21
Q

primers are

A

Short sequences of chemically synthesized DNA (10-30
bases) that Bind to the single stranded template DNA (or RNA)

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22
Q

how short are primers

A

10-30 bases

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23
Q

what are the primers used in PCR

A

forward (F) and reverse (R) primers

F & R primers flank and define the target DNA (gene) to
be copied

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24
Q

function of primers in PCR

A

Forward & Reverse primers flank and define the target DNA (gene) to be copied and Serve as recognition sites for the polymerase

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25
what serve as recognition sites for the polymerase
primers
26
polymerase enzymes are
Thermostable enzymes which synthesizes new DNA
27
functions of polymerase enzymes
thermostable enzymes that synthesizes new DNA and Recognizes primers as start point for synthesis they Synthesizes new strand in 5’ to 3’ direction
28
what direction do polymerase enzymes synthesizes new strands in
Synthesizes new strand in 5’ to 3’ direction
29
polymerase enzymes requires what to function
MgCL2 and PCR buffer
30
PCR reaction buffer generally contains
0-50mM Tris-HCl pH 8.3 up to 50mM KCl BSA
31
how much Tris-HCl does the PCR buffer contain
0-50mM Tris-HCl pH 8.3
32
function of PCR reaction buffer
Stabilize polymerase and the amplicons during PCR process
33
function of MgCL2
polymerase functioning
34
Thermal cycler/PCR Machine uses
heat to melt (separate) template DNA and anneal primers to template DNA Electronically controlled, capable of changing temperatures quickly and in a cyclic manner
35
What are the protocols for extracting genomic DNA
DNA should not be contaminated with spurious DNA, carbohydrates, phenols, or other compounds as these would inhibit DNA amplification
36
formula for typical PCR mix
C1V1 = C2V2
37
C1 is
concentration/amount at the start
38
C2 is the
concentration amount FINAL
39
V1 is
the volume START
40
V2 is the
final volume
41
PCR occurs in a 3 step process called a cycle what are the 3 steps
1. denaturation 2. annealing 3. elongation
42
what happens in Denaturation
Occurs at approx. 90-95C, 1min heatingseperates the double stranded DNA into single stranded DNA and this single stranded DNA serves as template for DNA cloning Heating separates the double stranded DNA = Denaturation Slow cooling anneals the two strands = Renaturation
43
denaturation occurs at what temperature
90-95C for 1 minute
44
in denaturation The H-bonds in isolated DNA is denatured & made single stranded and the ss DNA serves a
template for DNA cloning
45
Denaturation is when
Heating separates the double stranded DNA
46
Renaturation is when
Slow cooling anneals the two strands
47
how long does denaturation take
1 minute
48
what happens during Annealing in PCR reactions
Temperature quickly reduced to about 40-65C When primers to bind to complimentary regions at the beginning and end of target sequence of target DNA in single stranded DNA 45s 2 primers required, 1 for each strand Annealing temp. dependent on the length & base sequence of primer Allows primer to bind to template at high specificity
49
what is the temperature during annealing in PCR reactions
40-65C
50
annealing allows primers to
bind to complimentary regions at the beginning and end of target sequence of target DNA in single stranded DNA Allows primer to bind to template at high specificity
51
how long does annealing take in PCR reactions
45 seconds
51
how many primers required for annealing
2 primers 1 for each strand
51
temperature of elongation
70-75C (optimum temp for taq polymerase activity)
51
What happens during Elongation in a PCR reaction
Taq recognises primer and synthesises complimentary strand using dNTPs 70-75C optimum, temp for taq polymerase activity 2min
52
annealing is dependent on
temperature dependent on the length & base sequence of primer
52
PCR Fragments are resolved on either
agarose/ polyacrylamide gels
52
How long does elongation take
2 minutes
52
30 cycles of PCR can generate how many copies of the gene
over 1 billion copies
52
3 categories of applications of PCR
molecular identification sequencing genetic engineering
52
applications of pcr in molecular identification, sequencing and genetic engineering
DNA fingerprinting genotyping Mutation detection Paternity testing Sex determination Detection of pathogens Bioinformatics Genomic cloning Genome Projects gene expression studies
53
at least how many cycles are done in a PCR reaction
at least 30 cycles each cycle increases the copy number exponentially
53
explain how Primers being necessary is a limitation in PCR reactions
To synthesize primers, knowledge of the DNA sequence of interest must be known PCR cannot be used to study/amplify fragments of genes never studied before or be used in areas of the genome where no sequence info is available *Exception- Arbitrarily Primed PCR (AP-PCR), RAPDs or PCR where small known sequences are added to fragments to which primers are then made – AFLP
53
Limitations of PCR
Primers are necessary Size of amplified Fragment (amplificon)
53
To synthesize primers, knowledge of
the DNA sequence of interest must be known PCR cannot be used to study/amplify fragments of genes never studied before or be used in areas of the genome where no sequence info is available
53
explain how Size of amplified Fragment (amplificon) is a limitation in PCR reactions
PCR can clone fragments up to 5 kb without much difficulty, 40kb with some modification Some applications such as genome sequencing requires fragments>100kb to be amplified, therefore PCR cannot be used
53
PCR can clone fragments up to what kb without difficulty?
5 kb without much difficulty, 40kb with some modification Some applications such as genome sequencing requires fragments>100kb to be amplified, therefore PCR cannot be used
53
diagram PCR temperature profile

Principles Of molecular Biogy (3 decks)

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