PL3

Question 1

What does PCR do

Answer 1

Amplifies a specific DNA sequence which can be part of a complex mixture to possibly manipulate it

Question 2

What are some uses of PCR

Answer 2

Sequencing, DNA cloning, detection of pathogens, gene editing

Question 3

What does PCR generally depend on and why

Answer 3

Knowing the nucleotide sequences at the ends of the region to be amplified because it relies on producing primer sequences complementary and specific to ends

Question 4

What does a single reaction tube contain in PCR

Answer 4

DNA template
DNA polymerase stable at high temp
Primers complementary to each end of region to be amplified
DNTPs (monomers synthesized into amplified DNA)

Question 5

What does temp of tube depend on

Answer 5

Amount of GC pairs

Question 6

What does heat destable

Answer 6

Double helix (denatures)

Question 7

What is the repetition that is done many times in PCR (3 step cycle)

Answer 7

Denaturation, annealing of primers and extension by DNA polymerase

Question 8

What is the denaturation step of PCR

Answer 8

temp increased to near boiling to denature and separate DNA strands

Question 9

What is the annealing step of PCR

Answer 9

Temp decreased depending on sequence of primer to allow primers to base pair to complementary DNA template

Question 10

What is the extension step of PCR

Answer 10

Increased temp, polymerase has thermal resistance, polymerase extends primer to form nascent DNA strand

Question 11

What polymerase works best a high temps for PCR

Answer 11

Tac polymerase, also prevents reannealing

Question 12

Why must polymerase be heat resistant

Answer 12

Because we need to use high temps to denature DNA but we don’t want polymerase to fall apart as well because then we would have to add polymerase to each cycle (tiresome)

Question 13

What is polymerase Taq similar to and why

Answer 13

It does not have proofreading activity, more like pol alpha than pol s or e. Likely to make errors

Question 14

What does exponential amplification allow for

Answer 14

very sensitive detection of a DNA sequence in the sample and enables purification of substantial amounts of a specific DNA fragment for further use

Question 15

What is the goal of PCR

Answer 15

Exponential expansion of DNA

Question 16

Why can’t PCR be done forever in practice?

Answer 16

Limit of 35-40 times because primer will hybridize to places you don’t want it to, aka reaction makes mistakes

Question 17

What often happens to DNA fragments produced by PCR

Answer 17

They are often analyzed by gel electrophoresis

Question 18

What are the steps for gel electrophoresis

Answer 18

DNA restriction fragments placed in meshwork of gel (polyacrylamide or agarose)

Apply electric field

Molecules move through pores according to size

Shine it with something you can see

Question 19

What do all 4 tubes contain in DNA sequencing

Answer 19

DNA polymerase
Oligonucleotide primer
DNA template
dNTPs

Question 20

How to detect DNA in gel electrophoresis

Answer 20

Soak gel in a solution of a molecule that can interpolate into DNA and also glow when you shine UV on it

Question 21

What does each tube contain (different) in DNA sequencing

Answer 21

ddATP, ddGTP, ddTTP, ddCTP (chain terminators) low concentration

Question 22

What does oligonucleotide primer do

Answer 22

Anneals to one end of fragment you are trying to sequence (make a new strand)

Question 23

Does the DNA polymerase in DNA sequencing need to be heat resistant

Answer 23

No

Question 24

How do the chain terminators (ddNTPs) differ from the dNTPs

Answer 24

Has no oxygen on 3’, just hydrogen

Question 25

What does it mean from ddNTP’s not to have 3’ OH for DNA polymerase

Answer 25

Can’t add anything to it, chain ends when it gets incorporated

Question 26

What happens when there is ddGTP

Answer 26

When template has a C and G will come in and either there will be a G that furthers synthesis or a ddGTP that ends it

Question 27

What are the limitations of Sanger (DNA) sequencing

Answer 27

Polymerase only runs for 300-500 nucleotides and gels can only clearly resolve this much, so many separate individual sequencing reactions must be run to sequence a large region (hard to see difference between 512 and 513 vs 24 and 25)

Rate of sequence production is limited by total number of reactions that can be performed at a time

Question 28

Why is next generation sequencing such a breakthrough

Answer 28

Involved the development of new methods to allow a single sequencing machine to carry out millions of sequencing reactions simultaneously

Question 29

What is a type of NGS technology

Answer 29

Ligating the same linkers to a mixture of DNA fragments

Question 30

What happens after we’ve ligated the same linkers to a mixture of DNA fragments

Answer 30

DNA is then denatured and anneal it to a primer that is complementary and anchored to a solid support

Solid support undergoes PCR reaction so that you amplify DNA sequences but the sequences remain fixed in place

Question 31

What happens when sequence has been amplified using PCR in the NGS ligation of the same linkers

Answer 31

DS DNA is cute and only one strand is amplified with fluorescently labelled dNTPs (different colour for each base)

The it’s imaged and washed out and this is repeated

Question 32

What is the difference between the old way of assembling genome sequences vs the new way

Answer 32

Old = too much work, created aligned library, ordered sequences that were dictated by clone map
New: DNA is put into sequencing machine randomly, computational machines allows for the sequencing of that information

Question 33

What do the newest sequencing approaches skip

Answer 33

PCR: single molecules rather than clusters of molecules are being sequenced

Question 34

Nanopore steps

Answer 34

DNA is denatured
Interacts with motor proteins (moves through)
Moves through the motor protein’s electric current
Current depending on which base pair moves through

Question 35

What are motor proteins

Answer 35

Proteins that can move along a substrate or DNA molecule

Question 36

Advantages of nanopore

Answer 36

very long reads can be generated (many bases can be read)
Solves problem of genome assembly (easier to assemble repetitive DNA)
Very portable machine