L8- bisulphate sequencing, ChIP-seq + 3C

Question 1

Describe methylation (draw if poss)

Answer 1

Most common on cytosine
Hydrogen on C5 changed to CH3 group

Question 2

Why is methylation important in gene silencing?

Answer 2

Methylation used in gene silencing
1. X inactivation for one of the XX chromosomes to prevent double the needed proteins expressed by X
2. Germline gene silencing
3. Repeat region silencing

Question 3

Describe imprinting and what happens when it goes wrong

Answer 3

Way of distinguishing from maternal and paternal genes through methylation
Angelman and Prader-Willi Syndrome associated with chromosome 15 deletions
Which syndrome arises depends on if deletion is on maternal or paternal C15

Question 4

Why is methylation important in bacteria?

Answer 4

Distinguish self from non- self DNA
Allows digestion of foreign DNA (like phage)

Question 5

Why does CPiG show more methylation than CHG or CHH?

Answer 5

CG = palindromic, methylation will occur on opposite strand as well therefore methylation will be present after DNA replication
CHG and CHH will only have 1 methylation, therefore won’t be present after DNA replication and only 1 daughter cell will have methylation

Question 6

Why is CPiG under represented in the genome? What is the exception in CPiG?

Answer 6

• Methylated cytosine easily mutates to thymine then uracil through deamination so CPiG not shown often
• Promoter regions often have CPiG islands where if methylated then gene not expressed/silenced

Question 7

How can cytosine be converted to uracil? How is this useful?

Answer 7

Sodium bisulphite used to convert cytosine to uracil
Methylated cytosine is not converted to uracil using this method- remains methylated
Can be used for sequencing

Question 8

Explain Reduced Representation Bisulphite sequencing (RRBS)

Answer 8

Purification of genomic DNA -> use restriction enzyme that recognise + cut CCGG region -> repair the ends -> add A/T tails -> add adapters -> use size selection -> do bisulphite conversion (methylated stay methylated) -> PCR amplification -> sequencing
> used to analyse genome wide methylation levels on nucleotide level

Question 9

Explain PacBio sequencing and how it detects methylation

Answer 9

1. Immobolised DNA polymerase at bottom of well, detecting fluorescent nucleotides incorporated
2. Methylation gets in the way of polymerase so there is a pause before a base is incorporated
3. All PacBio sequencing can detect methylation

Question 10

Explain how Oxford Nanopore can detect methylation

Answer 10

1. Artificial membrane with pore protein embedded and a motor protein that feeds through single strand DNA
2. Characteristic shift in membrane electrical potential (mem. = electrically insulating)
3. Different signal with methylated base

Question 11

Explain chromatin immunoprecipitation (ChIP)

Answer 11

Used to isolate DNA bound to specific protein
1. Proteins covalently cross linked to DNA by treating with formaldehyde
2. Chromatin sheared by sonication or with exonuclease (ChIP-exo allows bound DNA to be trimmed to a binding site)
3. Immunoprecipitation and purification of bound DNA using antibody specific to protein of interest
Used to use microarrays but now use ChIP-seq

Question 12

Explain ChIP-seq

Answer 12

Instead of using microarrays is more common to use high throughput sequencing like illumina
Sequence the ChIP purified binding sites with illumina
Reads mapped onto reference genome and peaks show binding sites

Question 13

Explain Chromosome Conformation Capture (3C/4C/5C/Hi-C)

Answer 13

Basic steps: Used to detect interactions between DNA
1. Use formaldehyde to crosslink to strands of DNA
2. Restriction digest to cut off unwanted ends
3. Ligate the ends on one side (fish shape <><)
4. remove cross link and left with 2 halves of DNA from 2 different strands