Lab #10: DNA Extraction + PCR Flashcards
What are some of the cellular barriers presented to isolating DNA?
Cell wall
Cell membrane
Nuclear membrane
DNA packaging proteins
Other organelles
Other macromolecules
ETC.
How was DNA extracted from maize plants?
Using a series of these 3 processes:
1) Homogenization (chemical + mechanical)
2) Heat treatment
3) Centrifugation
How was homogenization of leaf sample carried out?
Mainly mechanically with a PESTLE in the presence of a chemical detergent, SDS (along with heat)
What are the components of Edward’s Buffer?
1) Tris Buffer (pH = 8)
2) SDS
3) EDTA
4) NaCl
What is the role of tris buffer in Edward’s Buffer?
Maintains pH of the solution around 8 to stabilize DNA molecules
What occurs to DNA if extracted in an acidic environment?
Risk of Depurination (loss of purine bases A + G) which would alter the DNA sequencing results!
What does SDS do?
SDS is an anionic or negatively charged detergent which breaks down the lipid membranes and denatures cellular proteins
Why was the sample heat treated AFTER mechanical homogenization?
To further denature cellular proteins; heat + SDS = almost full denaturation of proteins
What does EDTA do?
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!)
What does SDS stand for?
Sodium Dodecyl Sulfate
What does EDTA stand for?
Ethylenediaminetetraacetic acid
What is the cofactor for DNAses
Mg2+ and Mn2+
What does the heat treatment at 100 C do to the sample?
1) Further denatures proteins
2) Further solubilizes membranes
After homogenization + heat treatment, what did we do?
What was collected?
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
What does SDS do to membranes?
Solubilizes them by forming MICELLES
–> SDS inserts its hydrophobic tail into the membrane = organizational disruption
–> Causes phospholipids to take on micelle conformation!
After the first centrifugation and isolation of supernatant, what was done?
Addition of ISOPROPANOL (an alcohol) to the supernatant
Why was isopropanol added to the samples?
(general reason)
To initiate DNA precipitation
–> To “salt” the DNA out of solution
What charge does DNA have?
Negative
What is the role of NaCl in Edward’s buffer?
It neutralizes the charge of DNA = becomes less soluble in water (because it’s less charged) = Precipitation out of solution!!
Why did the isopropanol initiate DNA precipitation?
Because it disrupted the “hydration shell” around DNA
–> Broke the strong interactions between water and DNA, allowing for Na+ interact with DNA instead!
Isopropanol disrupts…
the hydrophilic interaction of water with the DNA phosphate backbone
What gives DNA its (-) charge?
Phosphate backbone!
What is a hydration shell?
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)
Upon removal of the hydration shell, what occurs to the DNA?
Interacts with Na+ = becomes less charged = precipitates out of solution!
After the addition of isopropanol, what was done?
Centrifugation!
–> Pellet was collected which contained DNA, salt, and RNA/traces of other contaminants
After collecting the (mostly) DNA pellet, what did we do?
Why?
Resuspended the pellet!
–> In tris-EDTA buffer with added RNAse A = to break up RNA into small fragments
PCR =
Polymerase Chain Reaction
PCR allows us to…
amplify (produce many copies) of a specific region of a DNA molecule
DNA polymerase
Enzyme that builds DNA strands but connecting dNTPs together utilizing a template strand as reference
What are the building blocks used by DNA polymerase?
dNTPS = deoxyribonucleotides
What is the larger 3D structure of DNA?
Double Helix!
How does DNA polymerase “know” what dNTP to add?
Complementary base pairing with the template strand
In what direction does DNA polymerase move?
(template and new strand reference
Synthesizes DNA from 5’ to 3’ end of NEW STRAND
Moves 3’ to 5’ along TEMPLATE STRAND
New DNA strands form _________ to __________
5’ to 3’
What is at the 3’ end of DNA?
Free OH group
What is at the 5’ end of DNA?
Free phosphate group
DNA polymerase needs a free __________ to initiate DNA synthesis
A free 3’-OH end!
What does DNA polymerase need to start adding base pairs?
a PRIMER
–> A small stretch of single-stranded, complementary DNA with a free 3’OH end
What is a primer?
a small stretch of single-stranded, complementary DNA with a free 3’OH end
Primers used in PCR differ by…
The direction they go in = the strand they associate with!
Forward Primer
Binds to the beginning of the desired gene at the 3’OH end of one strand of DNA
(Primer is 5’ to 3’)
Reverse Primer
Binds to the end of the desired gene at the 3’OH end of the complementary strand of DNA
Primers always… (2)
1) Bind to the 3’ end of a template strand!!
2) Go 5’ to 3’
What type of DNA polymerase is used in PCR?
WHY?
Taq polymerase (bacterial)
Because it functions at high temperatures (doesn’t denature) which are needed for PCR to work
What are all the components DNA polymerase needs to work? (3)
1) Primer
2) Mg2+ cofactor
3) dNTPs
What is the role of Mg2+ in DNA synthesis?
Serves as a cofactor for DNA polymerase
Where does the energy come from for the addition of Nucleotides to a strand?
The hydrolysis of 2 phosphate groups from the dNTPs
What are the 3 steps of PCR?
1) Denaturation
2) Annealing
3) Elongation/Extension
What temperatures are each stage of PCR carried out at?
1) Denaturation = ~95C
2) Annealing = ~55-65C
3) Elongation = ~72C
What is in a PCR tube when placed into thermocycler?
1) DNA
2) dNTPs
3) Taq polymerase
4) Mg2+
5) Forward + Reverse Primers
6) Buffer (Containing DMSO in our case)
Where does PCR take place?
Thermocycler
What is a thermocycler?
Machine that can rapidly and repetitively change its temperature over time
What is the denaturation step (PCR)?
A “short pulse” at 94C = Denatures DNA to separate the 2 strands into individua-single strands
What is the annealing step?
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
What is the elongation step?
Phase in which taq-polymerase is synthesizing new strands (starting from the primers)
What occurs directly after the primers anneal?
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
In PCR, you do not start producing complete target copies (copies bound on both ends by the primers) until…
In the products of the second cycle
How do you determine how many DNA molecules you will have after PCR is run?
of DNA molecules after PCR =
(# of DNA molecules at START) * 2^(# of PCR cycles complete)
–> X2^n
In the second cycle, how many COMPLETE target copies are produced?
2 per starting molecule
For each round of PCR, what is the difference in terms of # of total DNA copies produced and # of target DNA copies produced?
of total DNA copies > # of Target DNA copies
What were the contents of PCR Mastermix (buffer)?
1) Forward and reverse primers
2) Buffer with Mg2+
3) dNTPs
4) Taq Polymerase
5) DMSO
How does Mg2+ impact DNA polymerase?
It binds interacts with the (-) phosphate backbones of DNA to shield the polymerase from these (-) charges
What does DMSO do to DNA?
Disrupts its secondary structure: breaks H-bonds between the paired bases of opposing strands
–> allows for more efficient strand separation in PCR
Which strand gets the forward vs reverse primer?
Forward Primer = attaches to strand reading 5’ to 3’ L to R
Reverse Primer = attaches to strand reading 3’ to 5’ L to R
