Analytical Technology

Question 1

What does Sanger Sequencing utilise to create differently sized nucleotide strands?

Answer 1

dATPs are used for chain extension
Fluorescently labelled ddATPs are used as terminator nucleotides as they don’t have an OH on the 3’ end

Question 2

How were DNA fragments cloned in second generation sequencing?

Answer 2

100-200bp fragments cloned into bacterial artificial chromosomes (BACs)

Question 3

What advantages did next generation sequencing have over sanger sequencing?

Answer 3

Lots of molecules could be sequenced at a time
Price per base dropped drastically
Fluorescent bases used
Bridge amplification for detecting the template molecule

Question 4

What sort of cloning did massively parallel sequencing use?

Answer 4

Bridge amplification
1. DNA fragments isothermally amplified
2. Oligos are added which hybridise with the lawn adaptor region
3. A complimentary strand is amplified through bridge amplification
4. Chain extension is prevented by reversible terminator nucleotides
5. The colour that fluoresces corresponds to the nucleotide

Question 5

What advantages do third gen sequencing have over illumina?

Answer 5

Single molecule sequencing
Real time sequencing
Ultra long read lengths
Identifying base modifications

Question 6

What disadvantages do third gen sequencing have over illumina?

Answer 6

More expensive per base
Higher error rate

Question 7

Outline PacBio sequencing

Answer 7

1. DNA is ligated into SMRT-bell adaptors
2. These are put into zero-mode waveguides (ZMWs)
3. Fluorescently labelled primers attach to the adaptors and go round the entire DNA, giving a circular consensus sequence
4. Fluorescence is detected from a single base and gives a pulse corresponding to the nucleotide

Question 8

Outline the GridIon machine mechanism

Answer 8

1. Motor protein unravels DNA and passes it through the pore into solution
2. Another molecule can the attach and give direct sequencing of DNA/RNA/protein

Question 9

How can the level of expression of a genome be quantified?

Answer 9

RT-qPCR:
1. RNA has cDNA synthesised from it with reverse transcriptase
2. DNA is amplified via PCR

Question 9

How can the level of expression of a genome be quantified?

Answer 9

RT-qPCR:
1. RNA has cDNA synthesised from it with reverse transcriptase
2. DNA is amplified via PCR

Question 10

What can be used to determine the genes being expressed?

Answer 10

Northern blotting:
1. mRNA is separated with electrophoresis
2. purified mRNA is transferred to a membrane
3. A labelled probe is used to find the transcript
(this is not very quantitative)

Question 11

What are some limitations of microarrays?

Answer 11

Low resolution sequencing as the probe may contain a similar sequence
Have to be predesigned, so we don’t know if any probes are missing
Not very quantitative as there is a limit to the amount of RNA that can hybridise in one spot

Question 12

How is DNA methylation on a genome found?

Answer 12

Bisulphate sequencing.
Sodium bisulphate converts unmethylated cytosine to uracil

Question 13

How is DNA fragmented for bisulfate conversion?

Answer 13

DNA is digested with a restriction enzyme MSP1 which recognises CCG

Question 14

How are DNA binding proteins bound to DNA detected?

Answer 14

Chromatin Immunoprecipitation:
1. Proteins are covalently cross-linked to DNA by formaldehyde
2. Chromatin is sheared by sonication or endonucleases. ChIP-exo trims DNA to the binding site
3. Bound DNA is purified and immunoprecipitated with specific antibodies, then put in a microarray

Question 15

How are RNA binding proteins bound to RNA detected?

Answer 15

CLIP-seq
RIP-seq

Question 16

What is used to identify long range chromosomal interactions?

Answer 16

3C: One enhancer to an interacting region
4C: One enhancer to al its interacting regions
5C: Many enhancers to many tagerts
Hi-C: All enhancers to all interacting targets

Question 17

How are essential genes found?

Answer 17

TraDIS:
Genes are inactivated with transposons
Chromosomal regions are sequenced outwards from inverted repeats

Question 18

What is used to find transcribed regions?

Answer 18

RNA-seq

Question 19

How are open chromatids found?

Answer 19

DNAse-seq and FAIRE-seq

Question 20

How are DNA satellites identified?

Answer 20

DNA fingerprinting:
1. Northern blot DNA
2. Minisatellite sequences are used as a probe
3. Two minisatellite sequences for each probe show up

Question 21

How are animal origins of replication found?

Answer 21

DNA is treated with BrdU (thymidine analogue)
Microarrays are used to find where origins of replication are

Question 22

How is amount of protein expressed and by how many cells found?

Answer 22

GFP promotor trap:
1. Genes from promotor libraries are plated
2. FACS separates cells by active/inactive promotor
3. Inactive promotor cells are put into the animal model and the active cells taken out are quantified

Question 23

Who is phenotypic screening done?

Answer 23

BioLog array

Question 24

How is reporter gene output assayed for known genes?

Answer 24

Single-copy reporter fusions
Multi-copy reporter fisions

Question 25

How is reporter gene output assayed for unknown genes?

Answer 25

Modified transposons
Promotor traps

Question 26

How are the insertion sites of transposon hybrids determined?

Answer 26

TMDH:
1. Transposon mutants are lysed open and digested into 60bp fragments
2. Labelled dNTPs are put into the reaction
3. T7 promotor is labelled red and SP6 labelled green
4. Run-offs are put onto a microarray to give a series of red and green RNAs, showing where the genome has inserted

Question 27

How can promotors be found?

Answer 27

Linker scanning mutagenesis:
Proteins that recognise RNA polymerase activity bind to a TATA box and two promotor-proximal regions on thymidise kinase

Question 28

How are regulatory regions in promotors found?

Answer 28

Deletion analysis:
Deletions done and activity measured to find how critical the sequence is
(same vibe as TraDIS)

Question 29

How are enhancers identified?

Answer 29

Enhancer trapping:
Reporter genes with weak promotors are randomly integrated into the genome with transposons
Clones showing upregulated expression are isolated and insertion sites are analysed by cloning and sequencing. These genes are close to an enhancer

Question 30

How is nucleosome density found?

Answer 30

Nuclease sensitivity assays:
Nucleosomes are digested by nucleases
DNA fragments are hybridised with probes to show the nucleosome density