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BIO 204 > Lab #10: DNA Extraction + PCR > Flashcards

Lab #10: DNA Extraction + PCR Flashcards

1
Q

What are some of the cellular barriers presented to isolating DNA?

A

Cell wall
Cell membrane
Nuclear membrane
DNA packaging proteins
Other organelles
Other macromolecules
ETC.

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2
Q

How was DNA extracted from maize plants?

A

Using a series of these 3 processes:
1) Homogenization (chemical + mechanical)
2) Heat treatment
3) Centrifugation

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3
Q

How was homogenization of leaf sample carried out?

A

Mainly mechanically with a PESTLE in the presence of a chemical detergent, SDS (along with heat)

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4
Q

What are the components of Edward’s Buffer?

A

1) Tris Buffer (pH = 8)
2) SDS
3) EDTA
4) NaCl

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5
Q

What is the role of tris buffer in Edward’s Buffer?

A

Maintains pH of the solution around 8 to stabilize DNA molecules

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6
Q

What occurs to DNA if extracted in an acidic environment?

A

Risk of Depurination (loss of purine bases A + G) which would alter the DNA sequencing results!

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7
Q

What does SDS do?

A

SDS is an anionic or negatively charged detergent which breaks down the lipid membranes and denatures cellular proteins

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8
Q

Why was the sample heat treated AFTER mechanical homogenization?

A

To further denature cellular proteins; heat + SDS = almost full denaturation of proteins

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9
Q

What does EDTA do?

A

Chelates or binds cations, like Mg2+ and Mn2+, that are usually required for the activity of DNAses

–> Makes these cofactors unavailable to activate DNAses! (preventing DNA degradation!)

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10
Q

What does SDS stand for?

A

Sodium Dodecyl Sulfate

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11
Q

What does EDTA stand for?

A

Ethylenediaminetetraacetic acid

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12
Q

What is the cofactor for DNAses

A

Mg2+ and Mn2+

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13
Q

What does the heat treatment at 100 C do to the sample?

A

1) Further denatures proteins
2) Further solubilizes membranes

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14
Q

After homogenization + heat treatment, what did we do?

What was collected?

A

Centrifuged the sample!

Yielded:

Pellet = larger, undissolved components (membrane/vesicle remnants, cell wall pieces, intact cells/organelles, etc.),

Supernatant = DNA, RNA, traces of other macromolecules, and buffer

–> Supernatant COLLECTED

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15
Q

What does SDS do to membranes?

A

Solubilizes them by forming MICELLES

–> SDS inserts its hydrophobic tail into the membrane = organizational disruption

–> Causes phospholipids to take on micelle conformation!

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16
Q

After the first centrifugation and isolation of supernatant, what was done?

A

Addition of ISOPROPANOL (an alcohol) to the supernatant

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17
Q

Why was isopropanol added to the samples?

(general reason)

A

To initiate DNA precipitation

–> To “salt” the DNA out of solution

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18
Q

What charge does DNA have?

A

Negative

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19
Q

What is the role of NaCl in Edward’s buffer?

A

It neutralizes the charge of DNA = becomes less soluble in water (because it’s less charged) = Precipitation out of solution!!

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20
Q

Why did the isopropanol initiate DNA precipitation?

A

Because it disrupted the “hydration shell” around DNA

–> Broke the strong interactions between water and DNA, allowing for Na+ interact with DNA instead!

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21
Q

Isopropanol disrupts…

A

the hydrophilic interaction of water with the DNA phosphate backbone

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22
Q

What gives DNA its (-) charge?

A

Phosphate backbone!

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23
Q

What is a hydration shell?

A

A three-dimensional sphere of water molecules that surrounds a molecule or ion in an aqueous solution

–> Due to intermolecular forces with water (dipole interactions)

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24
Q

Upon removal of the hydration shell, what occurs to the DNA?

A

Interacts with Na+ = becomes less charged = precipitates out of solution!

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25
Q

After the addition of isopropanol, what was done?

A

Centrifugation!

–> Pellet was collected which contained DNA, salt, and RNA/traces of other contaminants

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26
Q

After collecting the (mostly) DNA pellet, what did we do?

Why?

A

Resuspended the pellet!

–> In tris-EDTA buffer with added RNAse A = to break up RNA into small fragments

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27
Q

PCR =

A

Polymerase Chain Reaction

28
Q

PCR allows us to…

A

amplify (produce many copies) of a specific region of a DNA molecule

29
Q

DNA polymerase

A

Enzyme that builds DNA strands but connecting dNTPs together utilizing a template strand as reference

30
Q

What are the building blocks used by DNA polymerase?

A

dNTPS = deoxyribonucleotides

31
Q

What is the larger 3D structure of DNA?

A

Double Helix!

32
Q

How does DNA polymerase “know” what dNTP to add?

A

Complementary base pairing with the template strand

33
Q

In what direction does DNA polymerase move?
(template and new strand reference

A

Synthesizes DNA from 5’ to 3’ end of NEW STRAND

Moves 3’ to 5’ along TEMPLATE STRAND

34
Q

New DNA strands form _________ to __________

A

5’ to 3’

35
Q

What is at the 3’ end of DNA?

A

Free OH group

36
Q

What is at the 5’ end of DNA?

A

Free phosphate group

37
Q

DNA polymerase needs a free __________ to initiate DNA synthesis

A

A free 3’-OH end!

38
Q

What does DNA polymerase need to start adding base pairs?

A

a PRIMER

–> A small stretch of single-stranded, complementary DNA with a free 3’OH end

39
Q

What is a primer?

A

a small stretch of single-stranded, complementary DNA with a free 3’OH end

40
Q

Primers used in PCR differ by…

A

The direction they go in = the strand they associate with!

41
Q

Forward Primer

A

Binds to the beginning of the desired gene at the 3’OH end of one strand of DNA
(Primer is 5’ to 3’)

42
Q

Reverse Primer

A

Binds to the end of the desired gene at the 3’OH end of the complementary strand of DNA

43
Q

Primers always… (2)

A

1) Bind to the 3’ end of a template strand!!
2) Go 5’ to 3’

44
Q

What type of DNA polymerase is used in PCR?

WHY?

A

Taq polymerase (bacterial)

Because it functions at high temperatures (doesn’t denature) which are needed for PCR to work

45
Q

What are all the components DNA polymerase needs to work? (3)

A

1) Primer
2) Mg2+ cofactor
3) dNTPs

46
Q

What is the role of Mg2+ in DNA synthesis?

A

Serves as a cofactor for DNA polymerase

47
Q

Where does the energy come from for the addition of Nucleotides to a strand?

A

The hydrolysis of 2 phosphate groups from the dNTPs

48
Q

What are the 3 steps of PCR?

A

1) Denaturation
2) Annealing
3) Elongation/Extension

49
Q

What temperatures are each stage of PCR carried out at?

A

1) Denaturation = ~95C
2) Annealing = ~55-65C
3) Elongation = ~72C

50
Q

What is in a PCR tube when placed into thermocycler?

A

1) DNA
2) dNTPs
3) Taq polymerase
4) Mg2+
5) Forward + Reverse Primers
6) Buffer (Containing DMSO in our case)

51
Q

Where does PCR take place?

A

Thermocycler

52
Q

What is a thermocycler?

A

Machine that can rapidly and repetitively change its temperature over time

53
Q

What is the denaturation step (PCR)?

A

A “short pulse” at 94C = Denatures DNA to separate the 2 strands into individua-single strands

54
Q

What is the annealing step?

A

A “cool-down” phase in which the temperature is dropped to ~55-65C which allows the primers (F + R) to anneal to each individual strand present

–> Annealing = bind/base pair

55
Q

What is the elongation step?

A

Phase in which taq-polymerase is synthesizing new strands (starting from the primers)

56
Q

What occurs directly after the primers anneal?

A

Taq polymerase immediately binds to the primers and begins strand formation (though slow because temperature is still pretty low)

–> Occurs at 72C which is Taq Polymerase optimal temp

57
Q

In PCR, you do not start producing complete target copies (copies bound on both ends by the primers) until…

A

In the products of the second cycle

58
Q

How do you determine how many DNA molecules you will have after PCR is run?

A

of DNA molecules after PCR =

(# of DNA molecules at START) * 2^(# of PCR cycles complete)

–> X2^n

59
Q

In the second cycle, how many COMPLETE target copies are produced?

A

2 per starting molecule

60
Q

For each round of PCR, what is the difference in terms of # of total DNA copies produced and # of target DNA copies produced?

A

of total DNA copies > # of Target DNA copies

61
Q

What were the contents of PCR Mastermix (buffer)?

A

1) Forward and reverse primers
2) Buffer with Mg2+
3) dNTPs
4) Taq Polymerase
5) DMSO

62
Q

How does Mg2+ impact DNA polymerase?

A

It binds interacts with the (-) phosphate backbones of DNA to shield the polymerase from these (-) charges

63
Q

What does DMSO do to DNA?

A

Disrupts its secondary structure: breaks H-bonds between the paired bases of opposing strands

–> allows for more efficient strand separation in PCR

64
Q

Which strand gets the forward vs reverse primer?

A

Forward Primer = attaches to strand reading 5’ to 3’ L to R

Reverse Primer = attaches to strand reading 3’ to 5’ L to R

65
Q
A

BIO 204 (10 decks)

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