Week 22: (A) Introducing Sanger Sequencing, genome sequencing strategies, and next generation (Illumina) sequencing.

Question 1

What is Sanger sequencing ?

Answer 1

sequencing by synthesis method

Question 2

What do we need for Sanger sequencing?

Answer 2

sequencing primer, nucleotide to create new strand

template DNA, DNA polymerase, some form of detection method

Question 3

What is used in the radioactive dye determination sequence method?

Answer 3

each combination of 4 bases, (deoxyNTPS) combined with 1 dideoxynucleotides which would terminate the reaction.

Question 4

What methods did Sanger use to sequence?

Answer 4

dye termination sequencing (radioactive method)

Question 5

What is the ddNTP labeled with?

Answer 5

fluorophore, diff colour for each ddNTP

Question 6

What types of ddNTPs are there?

Answer 6

one for all 4 bases

A, G, T, C

Question 7

How do we detect where ddNTPs terminate the sequence?

Answer 7

capillary electrophoresis

Question 8

How are the sequences separated in capillary electrophoresis?

Answer 8

sample buffer (+ve change) 
runs along a narrow glass tube with gel in it.
runs to +ve buffer solution. 
as it runs to buffer solu, it passes through a laser which allows the fluorescent detector to detect what base it has been terminated at

Question 9

How can the Sanger method fail?

Answer 9

if we get impurities in the solution by sequencing 2 strands at once with the same primer
(will cute both sequences potentially at the same point and cause overlay)

Question 10

What needs to happen in order to minimise overlay of sequencing but not have to create many sequences, primers etc.?

Answer 10

1) split the genome into manageable chunks (billions to 40,000-200,000 bases)
2) work out which order they are in
3) get the DNA sequence of each chunk
4) put them together and we have a genome sequence.

Question 11

How do we split the genome into manageable chunks?

Answer 11

use restriction enzyme. find one that is rare cutting (every 50-200kb) cut up into smaller chunks

Question 12

How do we clone the chunks of DNA sequence?

Answer 12

clone into a BAC vector to give artificial chromosomes

i.e a plasmid that can replicate DNA sequences of that size faithfully

Question 13

What is the plasmid called that we replicate and amplify chunks of DNA in?

Answer 13

BAC vector

bacterial artificial chromosome

Question 14

What does transfect mean?

Answer 14

introduce (genetic material) by infecting a cell with free nucleic acid.

Question 15

What happens after the DNA chunk is inserted into BAC?

Answer 15

it is colonised on an agar plate,
colonies form (each one a diff chunk of the sequence)
put into wet and iron as a single culture
purified
analyses independently

Question 16

how do we work out which order the DNA genomic sequences are in?

Answer 16

carry out an enzyme digest
use the enzyme digest fingerprints (overlapping regions)
to work out the order
create a physical map

Question 17

What is it called when we create a physical map from the enzyme digest?

Answer 17

tiling paths

like a jigsaw

Question 18

What is the stage of getting the DNA sequence of each chunk called?

Answer 18

shotgun sequence a see blow the whole thing a part

Question 19

What do we do to the genomic sequence in shotgun sequencing?

Answer 19

fragment the genomic sequence into a suitable size ~1200bp

Question 20

Why do we fragment the genomic sequence into 1200bp?

Answer 20

as the total length is 1400 bp so we will get some overlap when we shotgun the sequence

Question 21

Where do we put the fragmented genomic DNA?

Answer 21

into a sequencing vector

Question 22

What type of plasmid do we insert the fragmented genomic sequence into?

Answer 22

sequencing vector 
normal plasmid 
flanking the site were we insert out DNA
The 2 sites which correspond to standard primer sites 
use the exact same primer

Question 23

what is a contiguous?

Answer 23

the sequence assembles without the overlaps

Question 24

What is the economic problem of Sanger sequencing?

Answer 24

slow an expensive

Question 25

What is the difference between Sanger sequencing and illumina sequencing?

Answer 25

sanger: sequence all bases in one molecule at a time
Illumina: sequence all molecules the same time

Question 26

What is the disadvantage to Illumina?

Answer 26

relatively short reads (19bp now up to 300bp)

expensive for single sequence

Question 27

How does illumine work?

Answer 27

sequence one base at a time in millions of sequences

Question 28

What are the steps in illumina sequencing?

Answer 28

spread DNA sequence on a microscope slide
really diluted,
amplify DNA by PCR to create a polony
use a common universal primer
can sequence one base and stop then sequence the next base and stop

Question 29

How do we dilute the DNA sequence for illumina?

Answer 29

take a scalpel and take a bit the DNA sequence at the appropriate band on an agarose gel, put in an epindorf tube.
needs it spread out to see

Question 30

What is shotgun sequencing?

Answer 30

The method involves breaking the genome into a collection of small DNA fragments that are sequenced individually.