Lectures 23, 24

Question 1

How are plasmids extracted?

Answer 1

A colony on a plate= one transformation event
Blue= empty vector
White= insert bearing clone
S3L23

Question 2

What are minipreps?

3 steps?

Answer 2

O/N culture pelleted to remove broth

1. Cells are resuspended (add some solutions, EDA protects from proteases and nucleases)
2. Cells are lysed (SDS unfolds proteins, breakdowns membranes, etc)
3. Solution is treated to precipitate debris (acidic potassium acetate if NaOH, not needed if sample is denatured by boiling)
   Then just spin out debris (plasmid remains in supernatant)100mmHg

Question 3

How do we get the plasmid to precipitate?

Answer 3

Salt added to supernatant (NaCl, etc)
1-3 volumes of alcohol added to solution (ethanol or isopropanol) displaces water (DNA less soluble)
Chill on ice (lowers solubility)
Spin in centrifuge to collect DNA
Wash with 70% ethanol
Dry and resuspend in H2O or TE

Question 4

What are 4 common modifications of plasmid precipitation?

Answer 4

Resuspend and lyse in one step (dirtier but faster)
Addition if lysozyme to lysis buffer for gram +
Remove contaminating proteins using phenol/chloroform extraction (slower but cleaner)
Resuspension of plasmid in buffer containing RNAse A (digest tRNA, rRNA, mRNA)

Question 5

What does larger initial DNA sources mean?

Answer 5

Larger initial DNA sources=more bands=more complexity

Question 6

What is polymerase chain reaction (PCR)?

Answer 6

Using heat stable enzyme (DNA polymerase) and short fragments of DNA as start sites we can make billions of copies of a DNA fragment from a single copy
Use other pieces of DNA to search given the facet that DNA can find complementary sequences
Make a copy of that region, and another and another

Question 7

What a re the components of PCR?

Answer 7

Template DNA
Oligonucleotide primers
Deoxyribonucleotide triphosphates (dNTP)- building blocks
Heat-stable DNA polymerase (Taq polymerase) - must survive all the heating and cooling
Suitable buffer solution

Question 8

What a re the steps of PCR?

Answer 8

1. Initial denaturation at 95C for 1-1- mins (more CG pairs= longer time needed)
2. Denaturation at 94-95C for 30-120 seconds
3. Annealing at 40-65C (Primer specific) for 30-120 seconds
4. Extension at 68-78C for N minutes, N= product length in kb
5. Repeat 2,3,4 for 30-40 cycles, hold at 4C
   Slides 15-18 L23

Question 9

How many more primers than templates do you start with?
How many more nucleotide molecules than template?
How many more enzyme molecules per template?

Answer 9

Start with 10 million times more primer than template
Start with 10 billion times nucleotide molecules than template, pool is not really depleted
Have 100 thousand enzyme molecules per initial template
Slides 4-5 L24

Question 10

What is the PCR polymerase Taq?

Answer 10

From thermus aquaticus or cloned enzyme expressed in eschericia coli
Optimal temp is 75-80C
Has ~10% activity at 37C
Half life at 95C is 40 mins
No 3’ to 5’ exonuclease activity so any misincorporated nucleotides are not removed
Frequently adds a single adenine (A) to the 3’ end of a molecule

Question 11

What is the PCR polymerase Pfu?

Answer 11

From pyrococcus furiosus
Similar temp optimum to Taq
Much more thermostable (half life around 18 hours at 95C)
Does contain 3’ to 5’ exonuclease activity to remove misincorporated nucleotides
Does not generate 3’ dA overhangs
Amplifies longer products better

Question 12

How is a primer designed?

Answer 12

1. Select region to be amplified
   A) usually a DNA sequence from sequencing you have done
   B) size of amplicon can vary from 100-10000 bases
2. Intelligently choose a pair of primers
   A) annealing temps +/- 2C
   B) ~50% C+G
   C) have a GC clamp (2-3/5 at 3’)
   D) doesn’t false prime
   E) doesn’t form hairpins
   F) doesn’t form primer-dimers

Question 13

What is Tm and annealing temperature?

Equation

Answer 13

Tm is the temp that a double stranded sequence of the primer would fully melt into single stranded molecules
Annealing temp os a PCR cycle is generally tested as 5 degrees Celsius below Tm
Tm=2C x (A+T) + 4C x (G+C)

Slide 10 L24

Question 14

What is positive and negative controls with PCR?

Answer 14

Positive controls vary, especially when troubleshooting a non-working PCR
(Every component can be faulty)

Negative control is usually the entire reaction with no template DNA
PCR is sensitive enough that you can amplify contaminating DNA from yourself, pipette, water, etc

Question 15

How can PCR be applied to identification?

Answer 15

Primers are designed to span genome regions that contain repeated sequences
The repeated sequences are chose because different repeat numbers are found in a population
While one region Amy have few possibilities, observing many regions at a time can create a unique combination
Slides 14-16 L24

Question 16

How can PCR be used to disease test?

Answer 16

Primers can be designed to give different sizes for functional/non functional genes
Primers can be designed to amplify only functional/non functional genes
Primers can be designed to give a product that is cut/not cut with a restriction enzyme to reflect presence of a disease gene
PCR can amplify a disease region to be used for sequencing

Question 17

How can PCR be used for mutagenesis?

Answer 17

A primer can be developed with a single nucleotide mismatch, or a small insertion/deletion
The amplified product is cloned into a vector (possibly for expression in target cells)
Slide 18 L24

Question 18

How can PCR be used for sequencing and RT-PCR?

Answer 18

Cycle sequencing copies only one strand, from one primer
Terminators in the dNTP mixture make variable length, colour-coded fragments

RT-PCR amplifies an RNA sequence after it is reversed transcribed into DNA
Reverse transcriptase makes a ssDNA
PCR amplifies the ssDNA

Question 19

What is quantitative PCR (qPCR)?

Answer 19

Specialized PCR machines with fluorescent detectors for each sample
Product is quantified by:
1. SyBr green dye: binds dsDNA and fluoresces
2. Primers that fluoresce when bound to template (allows for multiple colours)
Product to be quantified is compared against a known control

Question 20

What is sequence assembly in PCR?

Answer 20

Many primers are synthesized with significant overlap by inputting sequence into a computer program
Each piece extends off of each other piece
End primers are used to amplify only the correctly assembled piece
Slide 21 L24