Lecture 11-MT2

Question 1

What direction does the primer strand go in?

Answer 1

5’ to 3’

Question 2

What direction does the template strand go in?

Answer 2

3’ to 5’

Question 3

Which strand is complementary and has free 3’ OH?

Answer 3

primer strand

Question 4

What do we need for DNA replication?

Answer 4

-DNA
-DNA polymerase
-Enzymes to help DNA polymerase
-Primers
-dNTPs

Question 5

What does PCR need?

Answer 5

-DNA
-dNTPs
-DNA pol
-DNA primers (short, around 20 bp)

Question 6

What are PCR steps

Answer 6

1. Melting/denaturation (95 degrees celsius)
2. Annealing (around 60 degrees celsius)
3. Elongation (72 degrees celsius)
4. Repeat 20-30 times

Question 7

Why is denaturation at 95 degrees celsius?

Answer 7

This temp breaks down hydrogen bonds and separates 2 strands

Question 8

For the annealing step why does temperature vary and depend on bases?

Answer 8

Temperature for annealing depends on bases b/c
-C-G base pairing has more hydrogen bonds (3)
-A-T has less (2) bonds
-it depends what is in DNA, more heat to break down more bonds

Question 9

Why is elongation always at 72 degrees celsius?

Answer 9

Optimal temp for TAQ polymerase (polymerase used in PCR)

Question 10

What is the order of PCR?

Answer 10

Denaturation, annealing, elongation

Question 11

What is the average size of CRISPR locus?

Answer 11

800-2000 bp

Question 12

Where do primers bind in CRISPR locus

Answer 12

100 bp upstream and downstream

Question 13

How are DNA samples separated?

Answer 13

By size

Question 14

What is electrophoresis?

Answer 14

Separates DNA molecules on an electric field

Question 15

In electrophoresis chamber we load the DNA on…

Answer 15

The (-) pole since DNA is (-) it will be attracted to the (+) side & migrate through the gel & be separated by size

Question 16

True or false: large fragments move faster through gel

Answer 16

False
-small fragments go faster

Question 17

What does a bigger fragment size in gel indicate?

Answer 17

The bigger the size the more spacers it has

Question 18

What is used for Sanger sequencing?

Answer 18

-1 primer
-makes ssDNA pieces
-not exponential
-2 kinds of nucleotides (regular and ddNTPs)

Question 19

What are dideoxynucleotides? (ddNTPs)

Answer 19

Used in Sanger sequencing
- does not have 3’ OH so no more nucleotides can be added–>termination of primer chain extension

Question 20

Why do we amplify DNA sequences?

Answer 20

Eliminates noise so we can have correct fragments

Question 21

What is Sanger’s method?

Answer 21

-modified PCR
-Electrophoresis
-Sequence determination

Question 22

How is electrophoresis modified in Sanger’s method?

Answer 22

Electrophoresis uses a modified laser beam
-when fragment reaches beam a fluorescent color corresponds to base

Question 23

True or false: Sanger sequencing uses large fragments, such as over 1000 bp

Answer 23

False
-Sanger sequencing uses small regions of DNA (800-1000 bp)
-if larger than 1000 you can miss a part of sequence

Question 24

Do you expect the sizes of the CRISPR loci analyzed
through electrophoresis (bands) to correspond to the sizes
of the CRISPR loci’s sequences (the ones sequenced)

Answer 24

No
-If sequences are too large they will not appear