Genomic Analysis Flashcards
Define genomics
The study of all the nucleotide sequences including structural genes, regulatory sequences and non-coding DNA segments in the chromosomes of an organism
What part of the protein cycle does genomics look at
Just DNA
What part of the protein cycle does functional genomics look at
The characterization of protein-DNA interactions on the genome of an organism
Looks at - DNA, RNA, Proteins
Define structural genomics
the dissection of the architectural features of genes and chromosomes - how they’re packaged up and their location
Define comparative genomics
the evolutionary relationships between the genes and proteins of different species
What does epigenomics/epigenetics look at
DNA methylation patterns, imprinting and DNA packaging
Define pharmacogenomics
new biological targets and new ways to design drugs and vaccines using genes.
E.g viral knock in etc
What is a genome
The single nucleotide sequence of an organisms hereditary information (DNA in humans).
How many base pairs are in the human genome
3.2X10[11] -3.2 billion
What was the first RNA genome sequenced
Bacteriophage MS2 - 1976
What was the first DNA genome to be sequenced
Phage Phi-X174 - 1977
Small with only 11 genes - this is to get in and out infected cells ASAP (smash and grab approach).
What is the trend between genome size and the number of genes
There is no real trend
What 3 things did the human genome project discover
Large centromeres of unsequenced repetitive data
21,700 genes
Only 1.5% actually code for proteins, the lowest % of all organisms
Of the 98.5% of the genome that don’t code for proteins, what does the rest do
Introns
Regulatory sequences (promoters etc)
Unique non-coding DNA
Repetitive DNA
How do mutations help calculate the age of an organism and its divergence from the evolutionary tree
DNA incorporates mutations at roughly an equal rate - 10[-5]-10[-6] mutations per base pair per generation.
This can act as a molecular clock - more mutations means more divergence from a common ancestor therefore the “newer” the species. (e.g. humans have more mutations than dinosaurs).
How do you compare genomes
Use a genome browser - can compare many species DNA sequences with each other
Computer algorithms align the sequences and provide a visual output of how alike they are.
What can genome browsers help show
Areas of the genome that are conserved throughout time across species - meaning the regions that are highly similar must be important to survival (Evolutionary conserved regions -ECR)
These similar sites are assessed to look for transcription factor binding sites.
Why is functional genomics, the study of DNA-protein interaction, important
Mis-regulation of transcription factors will change gene activity (up/down/on or off) and ultimately lead to changed protein levels = disease
How is a gene turned on (gene regulation)
Dependent on surface receptors, these allow substrates to bind which triggers intracellular pathways to recruit RNA polymerase by binding to the enhancer and start transcription and translation to make a protein from the desired gene.
What is the role of an enhancer
Fine tunes the expression of a gene by turning its transcription activity up and down, dependent on the proteins that bind to that receptor
What is the role of a promoter
Turns gene on
How does DNA footprinting work (used for finding transcription factor binding sites)
Label a short sequence with a radiolabel/florescent label (32p) with the transcription factor on this sequence.
Mix these sequences with transcription factors
Cleave this mixed sample where it shall cut around the transcription factor due to not being able to get into cut it because it is bound to the transcription factor.
Control - Cut up the same bit of DNA without a transcription factor, will now be cut randomly and through the desired gene
Carry out gel electrophoresis - these fragments form a ladder based on size. Missing area where the gene should be (showing that the transcription factors do bind to your desired gene).
How does chIP seq work (chromatin immunoprecipitation sequencing) - used for finding transcription factor binding sites
Find binding sites using genetic browser (enhancer and promoter usually).
Treat with formaldehyde to fix tissue (covalent link between everything in the cell).
Ultrasonic waves to break open cells and smash the DNA into small fragments (500-1000BP)
Add antibody to bind to protein of interest
Magnetic beads coated in specific protein for antibody are fished out with magnet.
Wash beads (with antibody, transcription factor and DNA) to remove everything else
DNA is purified with chloroform extraction and used.
Pure DNA sample that used to be bound to TF is then sequenced.
Gives you billions of 50 base pair sequences
Computers take these genome parts and align them with human genome
Because antibodies have been used to find the DNA originally, these DNA sequences will be much more common than the other non-attached parts of DNA in that gene.
This large quantity of attachment DNA sections show the areas where TFs bind in the gene.
What two factors are vital for ChIP seq
Must have a completed genome as a reference to start with and reliable antibodies to bind to desired protein to fish it out.
