Lecture 9

Question 1

What is Renaturation?

Answer 1

When single stranded DNA can come together to form double stranded structure

Question 2

What is the speed of how fast denaturation propogates?

Answer 2

It is slow to start off but once it begins it is easy to continue

Question 3

What is a nucleation process?

Answer 3

In the process of renaturation, the initial base pair formations that occur

Question 4

What is the speed of the nucleation process?

Answer 4

It is quite slow

Question 5

What is the speed of renaturation after nucleation occurs?

Answer 5

It is fast

Question 6

What is the difference between denaturation in cells and in experiments?

Answer 6

In cells we never completely denature the entire DNA

Question 7

What does Renaturation require?

Answer 7

• Proper base matching

* Nucleation and zippering

Question 8

What is Tm?

Answer 8

The midpoint of the transition going from double stranded to single stranded

Question 9

How does AT and GC content affect the Tm?

Answer 9

High GC content results in an elevated Tm

Question 10

Why does high GC content result in a higher Tm?

Answer 10

GC pairs have stronger base stacking interactions than AT pairs (NOT hydrogen binding)

Question 11

How does more GC increase Tm?

Answer 11

Because GC increases base stacking

Question 12

Which regions of DNA are easier to be separated?

Answer 12

Regions with high AT content

Question 13

How does increasing or decreasing pH affect Tm?

Answer 13

It will lower the value of Tm by disrupting hydrogen bonding

Question 14

How can changing salt concentrations (like magnesium) affect Tm?

Answer 14

High salt concentrations with cations will interact with the negative backbones of DNA that repel each other and act as a buffer and allow the two strands to come closer to increase Tm

Question 15

How does Low salt affect Tm and why?

Answer 15

Low salt decreases Tm because it destabilizes the double helix

Question 16

How does increased salt affect Tm?

Answer 16

Increased salt elevates Tm and stabilizes the double helix

Question 17

How is Tm affected by the match of a hybrid structure of DNA strands?

Answer 17

If the match is relatively poor, Tm will decrease because it will be easier to break apart the strands. If the match is relatively good, Tm will increase because it will be more difficult to break the strands apart

Question 18

What is meant by the term favoring hybridization?

Answer 18

Favouring what will cause two strands to come closer together

Question 19

What would favour a higher Tm?

Answer 19

• Structures with higher GC content
• Higher salinity
• pH of 7

Question 20

What conditions will favour hybridization?

Answer 20

Higher Salinity

Question 21

What is a big difference in structure between DNA and RNA?

Answer 21

RNA forms more functional folded molecules

Question 22

What kind of base pairing does RNA have?

Answer 22

Intrastrand base pairing

Question 23

What kind of base pairing does DNA have?

Answer 23

Interstrand base pairing

Question 24

What kind of base pairing is seen in RNA?

Answer 24

• Watson crick BP (A/U; G/C)

* Non-watson Crick (G/U)

Question 25

What is RNA stabilized by?

Answer 25

The same forces as DNA

•Base stacking (hydrophobic/van der Waals interactions)

Question 26

Why can’t the A260/A280 ratio be used to detect RNA in a sample?

Answer 26

Because the ratio is about 2.1 for RNA so it is relatively close to 1.95 for pure DNA

Question 27

When can DNA and RNA form a double stranded helix?

Answer 27

When there are two complementary sequences like in transcription

Question 28

Why is the absorbance at 260 nm for RNA higher?

Answer 28

Because it starts of partially single stranded

Question 29

What is the Absorption at 260 of free nucleotides relative to double strand and single stranded DNA?

Answer 29

A260nm of free nucleotides > SS > DS