LECTURE 7 (sequencing)

Question 1

Historical perspective

Answer 1

60s - RNA is the first NA to be sequenced
70s - DNA is sequenced by sanger and maxam gilbert mechanisms
80s - modification of Sanger
90s - 1st human genome project
00s - human genome project finishes
10s - HTS (High Throughput seq) which allowsd the development of metagenomics.

Question 2

What is sequencing?

Answer 2

The way of characterizing a molecule either determining the a.a of a protein or the bases of a NA

Question 3

Describe Maxam-gilbert sequencing

Answer 3

We use 3 hazardous chemicals: Dymethil sulphate, hydrazine and piperdine.
DS: attacks purines
H: attacks pyrimidines

A: DS + piperdine
A & G: DS in formic Acid + piperdine

C: hydrazine + piperdine
C & T: hydrazine in NaCl + piperdine

overall: run on a gel and we have the whole seq

Question 4

Describe Sanger sequencing.

Answer 4

a.k.a dideoxy sequencing. DNAP is a key thing for this sequencing e.g sequenase.
You synthesize the molecule up to a specific point and they terminate at a point (with a dideoxynucleotide) that it’s identified by the terminator. you end up with different fragments of different sizes that can be run on a gel to determine the positions of the different nucleotides.

Question 5

What are dideoxynucleotides?

Answer 5

They’re chain-elongating inhibitors of DNAP used in sanger sequencing

Question 6

What is cycle sequencing?

Answer 6

the introduction of Taq polymerase in sanger sequencing in the 80s. It’s similar to PCR it works by cycles of sequencing however it’s not exponential and uses a single primer.
the results are shown in a virtual chromatograph which shows peak that show the intensity of the fluorescence. Can be done by robots, easy to use but primers can’t be seen in the virtual chromatograph.

Question 7

Which was the 1st bact whose genome was sequenced?

Answer 7

Xylella fastidiosa

Question 8

How many genomes are sequenced today?

Answer 8

10^4

Question 9

What is 1st generation sequencing?

Answer 9

Sanger and Maxam-Gilbert.

Question 10

What is second generation sequencing?

Answer 10

HTS; there are three companies
Pyro-sequencing: for reading long seq
Polony: reads millions of bases
Solexa: Illumina cheapest

Question 11

What is the market leader in sequencing?

Answer 11

Illumina sequencing.

Question 12

What are metagenomics, what revolutionized metagenomics?

Answer 12

HTS revolutionized metagenomics which is the analysis of neandertal sequences.

Question 13

Describe illumina sequencing (2nd generation sequencing)

Answer 13

It’s a type of 2nd generation sequencing therefore it’s a HTS. A flow cell is used.

Question 14

Can we see primers in the virtual chromatograph of cycle sequencing?

Answer 14

no

Question 15

Fluorescent dyes in sequencing are seen under laser or UV light?

Answer 15

laser light

Question 16

Are fluorescent dyes used in sequencing? what do they dye?

Answer 16

yes for dying dideoxy nucleotides and for labeling sequencing primers

Question 17

Can we do nowadays a whole genome sequencing? what do we need for it?

Answer 17

Yes with bioinformatics is becoming cheaper to do

Question 18

Which polymerases are used in sequencing? (4)

Answer 18

Taq polymerase, klenow fragment, RT and sequenase.

Question 19

Which three compounds are used for labeling NA

Answer 19

32P, 33P and 35S