MBB 267 Week 3: RMC 6 Flashcards
What are the functional elements in the human genome?
include long-range chromosomal interactions, regions of open chromatin, DNA methylation sites, transcription factor binding sites, enhancers, promoters and coding and non-coding transcribed regions
What is the ENCODE project?
ENCODE (the ENCyclopaedia Of DNA Elements) is a project aimed at identifying all of the functional elements in the human genome. The project is an example of “Big Science”, a huge collaboration many organisation and people.
What are the methods used by ENCODE?
The methods used during the ENCODE project included RNA-seq, 5C and ChIP-seq, RT-PCR, CLIP-seq, RIP-seq, DNase-seq, FAIRE-seq, ChIA-PET, methyl450k and WGBS
How were genes identified in the ENCODE project?
By RT-PCR or by computationally predicted
How were RNA-binding proteins identified in the ENCODE project?
CLIP-seq and RIP-seq are used to identify RNA-binding proteins
How were regions of open chromatin identified in the ENCODE project?
DNase-seq and FAIRE-seq are methods of identifying regions of open chromatin. DNase-seq exploits open chromatin’s hypersensitivity to DNase I digestion. FAIRE-seq uses formaldehyde crosslinking of DNA to nucleosomes, and purifies unbound DNA.
How were long-ranged chromosomal interactions identified in the ENCODE project?
Used ChIA-PET and 5c. ChIA-PET stands for Chromosome Interaction Analysis by Paired-End Tag sequencing. Like 5C it identifies long-range chromosomal interactions, but it does so through ChIP-seq analysis of DNA-nucleosome complexes rather than direct ligation of interacting DNA regions.
How was DNA methylation identified in the ENCODE project?
methyl450k is a microarray-based method of identifying DNA methylation
What is WGBS?
WGBS stands for whole genome bisulphite sequencing
What is bisulphite conversion?
Bisulphate conversion is a method that deaminate cystine residues. Methylated cytosine does not undergo this conversion, and remains unchanged. This is exploited in Bisulphite sequencing (BS-seq). Bisulphite treatment causes the unmethylated cytosines to be converted to uracil, which is read as thymine during sequencing. By sequencing and comparing both the treated and untreated samples, we can identify the methylated sites
What is the biological significance of having an evolutionary conserved sequence?
Can indicate a consereve function because:
- It is a common observation that exons are more conserved than introns between species
- This is because mutations are more likely to cause problems in exons
- More generally, functionally important regions of the genome tend to be evolutionarily conserved
What does TraDIS stand for?
TRAnsposon Directed Insertion-site Sequecning
What is a transposon?
Transposons are “jumping genes” which can move around the genome through a “cut and paste” mechanism. They consist of a transposase gene, flanked by inverted repeat sequences that are recognised by the encoded transposase.
How do transposons work?
The transposon can be introduced to a bacterial cell, for example on a plasmid. By supplying transposase we can induce it to transpose and insert into the bacterial genome at a random location.
- If a gene is disrupted by the transposon it will be inactivated
- If the disrupted gene is essential, the mutant will not survive
- If we make millions of mutants, then we will find transposons in every gene that it is possible to disrupt
- Genes without insertions are likely to be essential
How does TraDIS work?
It is a controlled way of adding transposons. we then map the sequences to the reference genome. The more mapped sequences, the more sure we are that the genes is non-essential. If there isnt any reads in a part of the sequence then that means that this gene is essential (as all the cells didnt survive)
