Sequencing

Question 1

What is DNA sequencing?

Answer 1

Analysis of DNA using molecular biology techniques

Question 2

What can go wrong with molecular cloning?

Answer 2

• empty vector
• incorrect recombinant DNA orientation

Question 3

Why is DNA sequencing useful?

Answer 3

Can check for mutations

Question 4

Describe the steps involved with getting DNA out of bacteria (mini-prep)

Answer 4

• grow bacteria
• break them open
• plasmids are small and compact
• DNA is big & tangled
• results in purified DNA

Question 5

What is a restriction digest?

Answer 5

Digest DNA with a restriction enzyme.

Question 6

What technique is used to separate DNA fragments based on their sizes?

Answer 6

DNA electrophoresis

Question 7

What gel is used in electrophoresis?

Answer 7

Agarose gel

Question 8

Why is ethidium bromide used in electrophoresis?

Answer 8

To visualise the DNA fragments under UV light, as it becomes fluorescent.

Question 9

What is the purpose of a DNA ladder?

Answer 9

DNA ladder is used to estimate size

Question 10

What charge electrode does the DNA move to?

Answer 10

DNA is negative so it moves towards the positive electrode.

Question 11

What moves faster through the agarose gel - small or large fragments?

Answer 11

Small fragments move faster, large fragments move slower

Question 12

What are 3 other uses of PCR?

Answer 12

• modern genetic fingerprinting
• identification of repeat expansions (certain diseases - e.g. Huntington disease)
• identification of genomic rearrangements (cancer)

Question 13

What is RFLP?

Answer 13

Restriction Fragment Length Polymorphism (RFLP) - this is where the number of repeats affecting the size of the restriction fragments.

Question 14

What is the specific type of sequencing, which is another phrase for DNA sequencing ?

Answer 14

Sanger sequencing - a.k.a the chain termination method

Question 15

What 2 things are needed to be known in order for sequencing to occur?

Answer 15

• How long a DNA molecule is (how many base pairs)
• What the last nucleotide is

Question 16

What is the difference between PCR & sequencing (think primers)?

Answer 16

Only one primer used in sequencing

Question 17

What 2 molecules are the main features of sequencing?

Answer 17

dNTPs - deoxynucleoside triphosphate
ddNTPs - dideoxynucleoside triphosphate

Question 18

What is the difference between dNTPs & ddNTPs?

Answer 18

ddNTPs don’t have an OH group at carbon 3.
- this means that a phosphodiester bond can’t form with the 5’ phosphate of the next nucleotide.
- this leads to the termination of the sequence.

Question 19

What are the 4 ddNTPs?

Answer 19

ddATP, ddCTP, ddTTP, ddGTP

Question 20

What nucleotide is at the end of all DNA fragments?

Answer 20

A

Question 21

How do you manage to create loads of different length fragments in DNA sequencing?

Answer 21

Include loads of different dNTPs & ddNTPS

Question 22

How does modern automated DNA sequencing take place?

Answer 22

One reaction take place containing all 4 ddNTPs, that are labelled with different fluorescent labels.

Question 23

What is the advantage of using modern sequencing techniques compared to previously used sanger sequencing?

Answer 23

• easy
• cheap
• quick