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BIOL 311 > Chapter 8 > Flashcards

Chapter 8 Flashcards

1
Q

What is PCR?

A

Polymerase Chain Reaction is a method of amplifying DNA and synthesizing more DNA.

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2
Q

Summarize the DNA synthesis or replication process?

A

To synthesize DNA a DNA polymerase adds deoxyribonucleoside triphosphates or dNTPs (dATP, dTTP, dGTP, dCTP) to the 3’ carbon of the growing chain and the energy to synthesize DNA is a result of cleaving off pyrophosphates to produce energy.

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3
Q

What is cDNA?

A

This is the DNA that forms from reverse transcriptase of mRNA.

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4
Q

What can be determined with a PCR test?

A

Determining parenting, diseases, and criminal DNA.

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5
Q

What is a reverse primer?

A

This is a DNA primer that binds to the end of DNA strand on the top strand and it must be complementary to the source DNA and the extension occurs from right to left.

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6
Q

What is a forward primer?

A

This is a DNA primer that binds to the beginnng of the DNA strand on the bottom of the strand and it must be complementary to the source DNA and the extension occurs from left to right.

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7
Q

What is the difference between the primers in PCR and the primers in DNA replication?

A

The PCR contains DNA primers and the DNA replication contained RNA primers.

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8
Q

In what direction is the primer synthesized?

A

5’ to 3’

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9
Q

How are the 3’ end of the reverse and forward primers related to each other?

A

The 3’ ends of the primers face towards each other.

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10
Q

What are the 4 necessary requirements for PCR to occur?

A

1.) Template DNA
2.) DNA primers
3.) dNTPs (dATP, dCTP, dGTP, dTTP)
4.) Taq polymerase

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11
Q

What is taq polymerase?

A

This is the DNA polymerase that was originally isolated from bacteria from a hotspring and these enzymes are active in hot and cold temperatures over multiple heating and cooling cycles.

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12
Q

What is the first step of PCR?

A

The 2 strands of the template DNA will be broken apart or denatured at 95°C by disrupting the hydrogen bonding.

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13
Q

What is the second step of PCR?

A

The DNA primers are bind at 55-66°C so they can join or anneal the template DNA complementary to the DNA strand.

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14
Q

What is the third step of PCR?

A

Then the taq polymerase will bind to the primer at 72°C where the nucleotides are added to the 3’ end of the primer and DNA is synthesized.

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15
Q

How many strands of DNA are synthesized after 30 cycles of PCR?

A

There are approximately 30 cycles in a PCR reaction and there will be 2^30 strands of DNA produced.

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16
Q

What determines the melting temperatures?

A

1.) G-C Richness
2.) The length of the primer

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17
Q

What is the melting temperature?

A

This is the temperature at which 50% of the primers are bound and 50% are not.

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18
Q

Why do longer primers lead to a higher melting temperature?

A

The primers when longer lead to more hydrogen bonding.

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19
Q

What is the relationship between the melting and annealing temperature?

A

The melting temperature is a reference and the annealing temperature will be lower so the primer can bind and not melt.

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20
Q

What happens as the cycles increase?

A

The gene of interest to be amplified is getting more specific and disregards the unnecessary fragments.

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21
Q

Are the primer sequences included in the PCR product?

A

Yes

22
Q

What is quantitative PCR or qPCR?

A

This method allows us to quantify the amount of DNA in the original sample and the flueorescent dye gets inserted into double-stranded DNA can be quantified based on it.

23
Q

What is the threshold of qPCR?

A

This is the point at which the reaction reaches fluorescent intensity and whichever sample reaches it first contains more DNA.

24
Q

What is gel electrophoresis?

A

This is a method of separating DNA molecules through a gel matrix and the DNA will be distinguished by size.

25
Q

What is Sanger sequencing?

A

In particular ‘dideoxy’ sequencing or Sanger sequencing this means we do not know the DNA makeup for instances the bases.

26
Q

How can we check that PCR occurred and the DNA was amplified?

A

Gel electrophoresis and staining for visualization.

27
Q

What is used to make the gels?

A

The agarose polymer which is used to make up seaweed and separates the DNA.

28
Q

What is the gel submerged in?

A

The buffer solution.

29
Q

Where is the negative charge applied?

A

To the top of the gel where the DNA is added to the wells.

30
Q

Where is the positive charge applied?

A

To the bottom of the gel where the DNA is added to the wells.

31
Q

Where does the DNA move?

A

To the bottom because DNA has an overall negative charge from the phosphates.

32
Q

How does the DNA molecules move through the well?

A

The power supply provides an electric current the DNA then travel through the channels.

33
Q

How do larger DNA molecules move?

A

They move less and remain near the top.

34
Q

How do smaller DNA molecules move?

A

They move more and remain near the bottom.

35
Q

What is cathode?

A

Negatively charged

36
Q

What is anode?

A

Positively charged

37
Q

How do we visualize the DNA?

A

Since DNA is colourless to see the DNA in the gel we add compound that bind to DNA and are fluorescent under UV light such as SYBRsafe.

38
Q

What is a molecular ladder?

A

These contains DNA pieces of known sizes to help determine the sizes of the DNA sample - since the size is known we use the molecular ladder to determine the length of the DNA samples.

39
Q

What are Sanger sequences used for?

A

These are used for sequences smaller DNA chromosomes not entire genomes.

40
Q

What are the steps of Sanger sequencing?

A

1.) Denaturation
2.) Annealing
3.) Synthesis

41
Q

How are Sanger sequencing and PCR similar?

A

1.) The steps of sequencing and PCR
2.) DNA template
3.) DNA polymerase

42
Q

How are Sanger sequencing and PCR different?

A

1.) PCR amplifies but sequencing does not to that extent
2.) Sequencing needs only one primer
3.) Sequencing requires ddNTPs and dNTPs

43
Q

What is a ddNTP or dideoxynucleoside triphosphate?

A

This is the same as a dNTP however the 3’ carbon lacks an OH and only has an H.

44
Q

What happens when a ddNTP is added?

A

The ddNTP can be added to a strand however the synthesis terminates after it is added because it lacks the 3’ OH.

45
Q

How did the original Sanger sequencing process occur
step 1?

A

There were 4 reactions each contained (DNA template, DNA polymerase, dNTPs, 1 primer) then in each tube a different ddNTP was added (ddATP, ddTTP, ddCTP, ddGTP).

46
Q

What was the ratio of ddNTP to dNTP?

A

1:300 - Therefore rarely what happens is DNA polymerase would occasionally add a ddNTP RANDOMLY and then the synthesis terminates.

47
Q

What happens as a result of the ddNTPs?

A

DNA fragments of different sizes form as a result.

48
Q

How do we analyze the results from Sanger sequencing?

A

To a special gel that has polyacrylamide instead of agarose which is more precise and it can resolve the 1 base pair that is different.

49
Q

How do we determine the sequence from the gel?

A

Since it outlines what the 1 base pair that is different we can look at the ddNTP at the ends that are different and determine the sequence of the NEW complement DNA.

50
Q

Is the sequence of the primer included in the gel?

A

No - that is our baseline and you would already know what that is because you order it.

51
Q

How does automated Sanger sequencing occur?

A

Each ddNTP is labeled with a different fluorescent marker so they can be visualized and a single reaction occurs. Also, a laser detects the different ddNTPs.

52
Q

If there is an “N” peak where there are 2 options what does that mean for the diploid organism?

A

That the organism is heterozygous.

BIOL 311 (12 decks)

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