MBB 267 Week 1: RMC 1

Question 1

What is Sanger sequencing?

Answer 1

Uses dideoxy-nucleotides as chain terminators to produce differently-sized products which can be separated on a gel to determine the sequence of a DNA molecule.

Question 2

Difference between ddNTP and dNTP.

Answer 2

ddNTP lacks the -OH group in the ring, whereas dNTP doesn’t so it can join the DNA chain extension.

Question 3

What is Dye-terminator sequencing?

Answer 3

Uses fluorescently labels in ddNTPs, so instead of running the sequence 4 times, the labels allows the whole process to be completed in a single capillary tube.

Question 4

Generic facts of the human genome.

Answer 4

Larger than 3 billion base pairs (haploid size), across 24 distinct chromosomes.
The genome is around 50% repetitive sequences, incl. terminal repeats, tandem repeats and interspersed repeats.

Question 5

The relationship between the Human Genome Project (HGP) and the International Human Genome Sequencing Consortium (IHGSC)?

Answer 5

HGP was undertaken by IHGSC, a multi-national collaborative group.

Question 6

About the HGP donors.

Answer 6

Donors were anonymous and so the “human genome sequence” does not represent any single individual.

Question 7

What is the approach used by IHGSC to sequence the whole human genome?

Answer 7

The sequencers cannot read an entire chromosome therefore:
Human genomic DNA is extracted.
The DNA is fragmented: physically (sonification), enzymatically (restriction enzymes) or chemically (heat and divalent cations)
The size needed for the fragment is 100-200kb.
Fragments cloned into Bacterial Artificial Chromosomes (BACs), which fits fragments of 100-200kb and up to 300kb: the plasmid contains sequence elements which tricks E.coli cells into replicating them during the cell cycle as if they were a native plasmid.

Question 8

Within the approach taken by IHGSC, how does BAC cloning amplifies DNA?

Answer 8

By producing a BAC library:
The E.coli cells grow into colonies which contain millions of copies of each BAC.
The individual colonies were picked from the plates and kept in separate tubes.
These tubes are the BAC library.

Question 9

Within the approach taken by IHGSC, how were genetic and physical maps linked?

Answer 9

These maps of the human genomes were used to sequence the genome in an ordered manner.

Question 10

What are genetic/ physical maps?

Answer 10

They are a set of landmarks across the genome that we know the sequence of.

Question 11

Within the approach taken by IHGSC, what do genetic maps use to sequence the genome?

Answer 11

They rely on recombination frequency between genetic markers.

Question 12

what do physical maps use to sequence the genome?

Answer 12

They use molecular biology methods such as restriction digests and FISH.

Question 13

“, what was one of the major achievements of the IHGSC?

Answer 13

Establishing a dense set of genetic markers across the human genome.

Question 14

how did IHGSC find the genetic insert in the chromosome?

Answer 14

We can link the inserts to our genetic/physical map, then we can test the clones for the presence of a particular marker sequence by PCR.

Question 15

what did IHGSC do when some clones did not have a marker sequence?

Answer 15

They identified clones which overlap a clone containing a marker using BAC and sanger sequencing:
They sequenced the end of the fragment in the BAC containing the marker.
They designed a PCR primer using the sequence they obtained previously.
They look for other BAC clones which contain the end sequence.
The overlapping BAC clones are likely to come from a neighbouring genetic locus.

Question 16

What is shotgun sequencing?

Answer 16

An approach to sequencing a large region of DNA, where many random fragments are sequenced and then assembled computationally.

Question 17

Who used the shotgun sequencing?

Answer 17

IHGSC, who applied this method to sequence individual BAC clones. And Celera, who used this approach to sequence the first bacterial genome (Haemophilus influenzae) and the whole sequence of the human genome.

Question 18

How were BAC clones shotgun sequenced?

Answer 18

The process:
The BAC clone DNA was fragmented into smaller 5-10kb fragments, and cloned into plasmid vectors.
Design primers for the vector (because we know the vector’s sequence) and sequence into the DNA insert.
We can build up a consensus sequence in the computer.

Question 19

Who was Celera Genomics?

Answer 19

It was a private company that threatened to patent the genome and commercialise it.

Question 20

Why was it good that there was competition between IHGSC and Celera?

Answer 20

Because the competition accelerated the HGS effort.

Question 21

What was Celera’s approach into sequencing the human genome?

Answer 21

The approach:
Celera used a Shotgun Whole Genome Sequencing (WGS) approach instead of creating BAC clone contigs (a contig is a set of overlapping DNA segments that together represent a consensus region of the genome.)
The genome was fragmented into much smaller fragments of 2-50 Kbp cloned into plasmids to create a library.
They created a consensus sequence ~5 fold coverage (this means that it was sequenced 5 times as a minimum amount.)

Question 22

Who won?

Answer 22

It was a draw. The two companies came into agreement to publish simultaneously in Nature and Science. Celera’s approach was cheaper but the company used data released by IHGSC so no fair competition.

Question 23

Why did the IHGSC use clone-by-clone and not a shotgun approach?

Answer 23

Because:
needed to prove it was feasible for a complex, repeat rich human genome
Government funders are risk adverse.
Celera did not have this problem.
Assembly was easier and could be performed with confidence.
Finishing (closing gaps in the draft genome) is easier with clone-by-clone as gaps can be targeted individually.
It is better suited to a diverse international consortium, as BAC contigs could be shared easily between members.
In the end they were wrong. Genomes are now routinely sequenced using massively parallel Next Generation sequencing technologies which employ a shotgun strategy.