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2.1 Bioinformatics > Fergus - Lecture 1 - part 2 > Flashcards

Fergus - Lecture 1 - part 2 Flashcards

1
Q

What are the components of a typical sequencing reaction?

A

DNA template

Synthetic olgionucleotide primers

dATP, dCTP, dGTP, dTTP

Dideoxynucleotide (deoxynucleotide with no O on 4’ C)

DNA polymerase

Buffer

Detection label

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2
Q

How does a sequencing reaction work?
(5)

A

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

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3
Q

Give some examples of DNA templates

A

Denatured double stranded templates

Cloning vectors with built in primer binding sites

PCR products

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4
Q

Learn vector in slides

A

Learn vector in slides

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5
Q

Why is DNA sizing needed?

A

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

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6
Q

What is shearing and how is it used?

A

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

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7
Q

What are the four ways of DNA sizing?

A

Random shearing and subcloning of DNA

Exonuclease III digestion of DNA

Progressive sequencing of DNA

Next generation sequencing

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8
Q

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

A

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

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9
Q

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

A

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

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10
Q

Write a note on dNTPs
(4)

A

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

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11
Q

Give an example of a nucleotide analogue

A

7-deaza dGTP

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12
Q

Write a note on ddNTP
(3)

A

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

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13
Q

Write a note on DNA polymeras

A

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

T7 (sequenase)

Taq polymerase

Thermosequenase

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14
Q

Name two radioactive labels

A

35S
33P

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15
Q

What type of electrophoresis is most common in automated sequencers?

A

Capillary electrophoresis

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16
Q

How does automated sequencing work?

A

Detection is done by incorporating fluorescent tags into primers

17
Q

List three different types of automated sequencers

A

ABI

Lycor

Megabase

18
Q

Write a note on ABI
(6)

A

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

19
Q

Write a note on Lycor
(4)

A

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

20
Q

Write a note on megabase

A

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

21
Q

How does big dye labelling work?
(3)

A

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

22
Q

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

A

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

23
Q

What is shotgunning?

A

Breaking apart a BAC into random fragments of different sizes

24
Q

What is whole-genome shotgunning?

A

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

25
Q

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

A

High resolution physical map required

Highly redundant clone library required

26
Q

What is next generation sequencing?

A

The ability to sequence whole genomes in a single run

27
Q

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

A

Roche 454

Illumina

Life technologies

28
Q

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

A

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

29
Q

What are the three types of new generation sequencing?

A

Whole genome sequencing

Whole exome sequencing

Targeted sequencing

30
Q

Write a note on whole genome sequencing
(5)

A

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

31
Q

Write a note on whole exome sequencing
(4)

A

Captures fragments related to genes

Can sequence up to 50 or 100 times depth

We detect all polymorphisms in protein coding regions

Cost effective

32
Q

Write a note on targeted sequencing
(4)

A

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

33
Q

Write a note on the roche system
(9)

A

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

34
Q

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

A

A chip is used instead of a bead

This leads to bridge amplification -> amplify the original fragment and copy it multiple times

35
Q

How is medial genetics different to other medical diagnostics?

A

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

36
Q

What is a pedigree analysis?

A

Whole family genetics

37
Q

Give some examples of human genome databases
(3)

A

NCBI -> National Centre for Biotechnology Information

Ensemble -> collab between European Bioinformatics Institute and Wellcome Trust

UCSC Genome browser (small academic group)

2.1 Bioinformatics (7 decks)

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