DNA Sequencing

Question 1

DNA Hybridisation

Answer 1

• when two ssDNA molecules join to form a dsDNA molecule
• this can only occur if the sequences are the complement of each other
• if only one or a few base pairs are mismatched then hybridisation can still occur, otherwise it willnot

Question 2

Spontaneous Hybridisation

Answer 2

-complementary sequences of ssDNA hybridise spontaneously below ~65-70’C

Question 3

Denaturing DNA

Answer 3

• heating dsDNA above a melting temperature Tm causes it to denature into ssDNA
• Tm depends on chain length, the longer the chain the higher Tm and base sequence, C-G rich regions have higher Tm since there are 3 hydrogen bonds between C and G but only 2 between A and T
• all DNA molecules melt ar T>95’C without significant hydrolysis of the sugar-phosphate backbone

Question 4

Polymerase Chain Reaction

Answer 4

-heat to 95’C, dsDNA denatures
-T=50-65’C ,primers anneal, you need to know the sequence of a short region at the end of the sample to create complementary primers
-T=75-80’C, Taq. DNA polymerase transcribes ssDNA into dsDNA
-this repeats on a cycle
-exponential amplification
no. of molecules = 2^n
n=number of rounds

Question 5

Sanger Sequencing

Answer 5

• PCR but with some ddNTP bases mixed I’m
• these bases terminate chain when added as they are missing the O required for the next base to be attached
• since the addition of these modified bases is random, over many cycles of PCR you end up with chains of all possible lengths each terminating with a tagged base
• perform gel electrophoresis on the fragments
• now that they are sorted into size order, the base sequence can be read off
• for large samples, shotgun sequencing also has to be used as only 500-800 bps can be sequenced at a time with this method

Question 6

Problems With the Sanger Method

Answer 6

• time
• cost
• errors generated by amplifying DNA using PCR
• requires chemical labelling (fluorescence/radioactivity)

Question 7

Shotgun Sequencing

Answer 7

• larger samples are broken down into smaller overlapping fragments by DNA restriction enzymes
• then the sequence is put together at the end

Question 8

Ion Torrent Sequencing

Question 9

Using Nanotechnology to Sequence DNA

Concept

Answer 9

-radial confinement and electrical read out

Question 10

Using Nanotechnology to Sequence DNA

Potential Advantages

Answer 10

• high sensitivity - single molecules
• long read lengths
• high fidelity?? (error rate?)
• spin-off ideas and techniques for other aspects of understanding biology

Question 11

What is the speed of DNA and RNA polymerases?

Answer 11

• RNA polymerase turns over 20 bases per second

- DNA polymerase is even faster

Question 12

Small Cantilever AFM Images

Answer 12

• a smaller cantilever has a higher resonance so can be scanned faster without resonating
• but still not fast enough to ‘see’ or ‘feel’ RNA polymerase motor action in enough detail to sequence DNA
• increasing scan speed also compromises spatial resolution

Question 13

Small Cantilever AFM Images vs Conventional AFM

Answer 13

• small cantilever: 10 frames/s

- conventional: 0.03 frames/s