Southern blotting, DNA sequencing

Question 1

What is meant by a DNA molecule denaturing?

Answer 1

Hydrogen bonds in base pairs are broken

DNA becomes single-stranded

Question 2

How is DNA denatured?

Answer 2

Heating it to high temperatures

Treated it with alkaline solution

Question 3

What is meant by DNA renaturing?

Answer 3

Hydrogen bonds in base pairs reform

form double-stranded DNA

Question 4

How is DNA renatured?

Answer 4

Cooling it down

Question 5

What is DNA hybridisation?

Answer 5

Add labelled DNA probe to single-stranded DNA that has complementary base sequence
DNA probe will bind to DNA

Question 6

What is a DNA probe?

Answer 6

Short single-stranded section of DNA

Question 7

How can DNA probes be labelled?

Answer 7

Radioactively

Fluorescent markers

Question 8

What is the purpose of DNA probes being labelled?

Answer 8

So can identify them bound to DNA

Question 9

What is the first step of Southern blotting?

Answer 9

DNA gel electrophoresis

Question 10

What is done to DNA gel electrophoresis results in Southern blotting?

Answer 10

Gel is soaked in alkaline solution

to denature DNA

Question 11

What is done to the denatured DNA on the gel electrophoresis results in Southerin blotting?

Answer 11

Single-stranded DNA fragments are moved to nitrocellulose paper by capillary action

Question 12

What is done to the nitrocellulose paper in Southern blotting?

Answer 12

Placed in solution containing labelled DNA probes

then washed off to remove unbound DNA probes

Question 13

What is done to identify the presence of DNA probes on the nitrocellulose paper?

Answer 13

Use photographic film if DNA probes are radioactively labelled

Fluorescent detection methods if DNA probes are fluorescently labelled

Question 14

Do DNA probes have a completely complementary base sequence to the target DNA sequence?

Answer 14

No

Question 15

What affects how tightly DNA probes bind to the target DNA sequence?

Answer 15

How complementary the DNA probes are to the DNA sequence - the more complementary, the more tightly they bind

Question 16

Do DNA probes have to completely align with the DNA target sequence?

Answer 16

No, only part of them has to bind

Question 17

What is DNA sequencing?

Answer 17

Process of working out base sequence of a DNA fragment

Question 18

How is a ddNTP different to a dNTP?

Answer 18

dNTP 3’ is bound to OH

ddNTP 3’ is bound it H only

Question 19

How does a ddNTP affect elongation of a DNA strand? Why?

Answer 19

DNA polymerase cannot add next nucleotide due to lack of 3’ OH
so elongation of DNA strand is terminated

Question 20

What are the requirements of DNA sequencing?

Answer 20

Four containers, each with

• DNA fragment to be sequenced
• DNA primer
• dNTPs
• DNA polymerase
• one type of ddNTP

Question 21

What is a condition of the DNA primer in DNA sequencing? Why?

Answer 21

Must have complementary base sequence to 3’ end of DNA fragment
since DNA polymerase will extend new DNA strand 5’ to 3’

Question 22

Does DNA polymerase add on a dNTP or a ddNTP to the new DNA strand in DNA sequencing?

Answer 22

Chance of either!

Question 23

What is done to the contents of the four containers after they’re incubated?

Answer 23

For each container, new DNA strands put into a lane

then DNA strands separated out by DNA gel electrophoresis

Question 24

How are the results of DNA sequencing interpreted?

Answer 24

Read off sequence by furthest band to closest band

complementary base sequence will be that of DNA fragment

Question 25

Why are the results of DNA sequencing read from furthest band to closest band?

Answer 25

Furthest band will be lightest, and hence shortest

so will be first base in DNA sequence

Question 26

What are the uses of Southern blotting?

Answer 26

Investigate gene structure

mutations