Fergus - Lecture 1 - part 2

Question 1

What are the components of a typical sequencing reaction?

Answer 1

DNA template

Synthetic olgionucleotide primers

dATP, dCTP, dGTP, dTTP

Dideoxynucleotide (deoxynucleotide with no O on 4’ C)

DNA polymerase

Buffer

Detection label

Question 2

How does a sequencing reaction work?
(5)

Answer 2

Denature double strand

Add mix of DNA nucleotides and dideoxynucleotides e.g. ddATP

Most of the time the DNA will add on a normal nucleotide but sometimes it will add on a ddNTP and wont be able to extend further and will result in a termination

However because there is no OH at the 3’ end they wont be able to extend

We can find the position of each ddNTP (ddATP, ddCTP etc) by finding out the sizes of all the terminated products using gel electrophoresis

KNOW DIAGRAM AND GEL

Question 3

Give some examples of DNA templates

Answer 3

Denatured double stranded templates

Cloning vectors with built in primer binding sites

PCR products

Question 4

Learn vector in slides

Answer 4

Learn vector in slides

Question 5

Why is DNA sizing needed?

Answer 5

If you have a 10,000 bp sequence you could design a primer for the first 1,000 bps and keep repeating this but it would takes months

Instead we randomly shear and subclone DNA

Question 6

What is shearing and how is it used?

Answer 6

Randomly physically chopping up the sequence into loads and loads of smaller overlapping fragments

We then clone these into vectors

We randomly sequence about 100 of these (on the one day)

There will be some overlap but you will have the whole sequence done in a day

Question 7

What are the four ways of DNA sizing?

Answer 7

Random shearing and subcloning of DNA

Exonuclease III digestion of DNA

Progressive sequencing of DNA

Next generation sequencing

Question 8

How does exonuclease III digestion work (in DNA sizing)?

Answer 8

KNOW DIAGRAM FROM LECTURE

Exonucleases A and B degrade DNA at different time points to give different sizes of sequences

There might be small amount of overlapping

Question 9

Write a note on olgionucleotides (in DNA sizing)
(3)

Answer 9

Act as the DNA polymerase priming site

Complementary to known sequence or to a universal primer site in a vector

Can be labelled with radioactive or fluorescent labels to enable detection of sequencing products

Question 10

Write a note on dNTPs
(4)

Answer 10

Components of DNA
Required by DNA polymerase for chain extension
Supplied at concentrations that will not limit DNA

Nucleotide analogues can be used to avoid DNA structure problems

Question 11

Give an example of a nucleotide analogue

Answer 11

7-deaza dGTP

Question 12

Write a note on ddNTP
(3)

Answer 12

Incorporated into extending DNA strand as normal
Once incorporated chain extension terminates
Can be labelled fluorescently for automated detection

Question 13

Write a note on DNA polymeras

Answer 13

Adds dNTPS or ddNTPS onto the 3’ hydroxyl of an extending strand

T7 (sequenase)

Taq polymerase

Thermosequenase

Question 14

Name two radioactive labels

Answer 14

35S
33P

Question 15

What type of electrophoresis is most common in automated sequencers?

Answer 15

Capillary electrophoresis

Question 16

How does automated sequencing work?

Answer 16

Detection is done by incorporating fluorescent tags into primers

Question 17

List three different types of automated sequencers

Answer 17

ABI

Lycor

Megabase

Question 18

Write a note on ABI
(6)

Answer 18

Most commonly used

Can use up to 4 fluorescent tags

Label is on the ddNTP

All 4 termination reactions can be loaded into the 1 well

Laser scans across gel with detection microscope

Capillary electrophoresis

Question 19

Write a note on Lycor
(4)

Answer 19

2 fluorecent tags
Label is on the primer
2 sequencing reactions (a forward and reverse) require 4 lanes
Dual laser scans across gel with detection microscope

Question 20

Write a note on megabase

Answer 20

4 fluorescent tags can be use
All 4 termination reactions can be loaded into 1 capillary

Question 21

How does big dye labelling work?
(3)

Answer 21

Each ddNTP is labelled with one of the four fluorescent dyes

The sequencer can determine which ddNTP terminated the reaction and therefore can determine which nucleotide base (A, T, G, C) is at that point

4 reactions can be performed in the one tube

Question 22

What was the sequencing strategy of the original human genome project?
(9)

Answer 22

It was done one chromosome at a time

They chopped the chromosome into chunks

These fragments were inserted into plasmids, bacterial artificial chromosomes (BAC) and cloned in bacteria

The fragments are then mapped so its known what region of the chromosome they came from

Each BAC insert is then sequenced

Each BAC is shotgunned into pieces, process repeated several times to give different sets of fragments

Fragments are cloned into small vectors and then sequenced

Sequences are fed into a computer which looks for overlaps

When enough of the fragments have been sequenced the sequence of the original BAC can be assembled -> this is done for all BACs to give a complete chromosomal sequence

Question 23

What is shotgunning?

Answer 23

Breaking apart a BAC into random fragments of different sizes

Question 24

What is whole-genome shotgunning?

Answer 24

This is when a whole genome is sequenced without the use of BACs i.e. the whole chromosome is shotgunned

Question 25

What are the main challenges for large scale sequencing? (2)

Answer 25

High resolution physical map required Highly redundant clone library required

Question 26

What is next generation sequencing?

Answer 26

The ability to sequence whole genomes in a single run

Question 27

What automation is used in next generation sequencing? (3)

Answer 27

Roche 454 Illumina Life technologies

Question 28

How does next generation sequencing work, what is it based on? (3)

Answer 28

Fragment a genome Separate the fragments on to some sort of solid support e.g. beads or chips We detect the termination products via fluorescent

Question 29

What are the three types of new generation sequencing?

Answer 29

Whole genome sequencing Whole exome sequencing Targeted sequencing

Question 30

Write a note on whole genome sequencing (5)

Answer 30

We will find everything including junk DNA 30x coverage (every base sequenced 30 times) Lots of overlap Lots of sequence variations e.g. deletions Very wasteful in diagnostic labs

Question 31

Write a note on whole exome sequencing (4)

Answer 31

Captures fragments related to genes Can sequence up to 50 or 100 times depth We detect all polymorphisms in protein coding regions Cost effective

Question 32

Write a note on targeted sequencing (4)

Answer 32

Targets fragments for individual disease Can sequence up to 500 times depth e.g. can look at just the exons of cystic fibrosis Most cost effective -> can do 50 genes in 8 individuals in one run

Question 33

Write a note on the roche system (9)

Answer 33

Amplification of fragments in an oil aqueous emulsion Fragment the genome Join adapters to end of fragments Mix fragments with excess micro beads -> only one bead binds to one fragment Mix beads with PCR reagent (=micro-reactors) Perform emulsion PCR Break down emulsion so we are left with only the amplified single fragment Pipette them into PicoTiter plate -> one bead per well Load with sequencing reaction

Question 34

How does the illumina system of whole genome sequencing work? (2)

Answer 34

A chip is used instead of a bead This leads to bridge amplification -> amplify the original fragment and copy it multiple times

Question 35

How is medial genetics different to other medical diagnostics?

Answer 35

You can tell someone that they will die of a disease before they even have symptoms

Question 36

What is a pedigree analysis?

Answer 36

Whole family genetics

Question 37

Give some examples of human genome databases (3)

Answer 37

NCBI -> National Centre for Biotechnology Information Ensemble -> collab between European Bioinformatics Institute and Wellcome Trust UCSC Genome browser (small academic group)