Describe the basic structure of DNA
Is the instruction manual for all living cells
Double stranded
Chain of nucleotides (A, C, G, T) joined to a sugar phosphate backbone
Arranged in a double helix
Pyramidines = C and T
Purines = A and G
Double helix arrangement -> unwinds when replication occurs
Describe how DNA replications (basic)
- each strand in the double helix acts as a template for synthesis of a new complementary strand
- hydrogen bonds break and double helix opens up
- replication occurs in 5->3 direction (primer binds at the 5’ end and new nucleotides added in the 3’ direction)
(called 5/3 as the ‘5’ end is where the 5th carbon in the ring has a phosphate group attached. ‘3’ end is where the hydroxyl group is attached on the third carbon in the ring)
- DNA polymerase adds complementary bases onwards from the primer
- (leading strand is synthesised continuously, and the lagging strand in fragments)
- DNA ligase is used to fill in any missing gaps in the newly synthesised strands)
Describe Sanger sequencing (what you need and how it works)
Developed by Frederick Sanger (1977) Ingredients: - DNA sample to be sequenced - primers - DNA polymerase enzyme - nucleotides (NTP's) - di-deoxynucleotides (ddNTP's) (100:1 ratio), fluorescently labelled
- First, DNA is denatured at 90 to separate strands
- Then, annealing of primers at 45 degrees
- Then, DNA polymerase extends the strands at 75 degrees and ddNTP’s are fluorescently labelled and incorporated at each position (due to ratio)
- Incorporation of ddNTP terminates the strand as it lacks phosphodiester bond for next nucleotide to bind to
Gel electrophoresis then carried out to arrange the segments into order, and fluorescence detected using computer and gives you the sequence
Describe NGS (Illumina sequencing) roughly:
1) sample prep:
- Fragmented DNA sample with millions of fragments added to a flow tray containing two types of fixed oligonucleotides
- Sample heated to denature the dsDNA into ssDNA
- ssDNA will bind to one type of the oligonucleotides on the tray
2) cluster formation
- DNA polymerase then added and makes bound ssDNA into dsDNA
- The new double strand bends like a bridge and binds to the second type of oligonucleotides fixed on the tray
- denaturing occurs again and the process is repeated
3) sequencing (fluorescence emitted is detected by computer to quantify how much DNA has been replicated in the clusters)
4) data analysis
- small fragments formed in the clusters are aligned next to the reference genome using a computer
Sanger sequencing may need to be used to sequence any virgin areas
Describe differences between Sanger sequencing and NGS sequencing
Sanger - good for first time sequencing
- slower
- more expensive
- longer reads (1000’s of bp’s long)
- more accurate
- single experiments
NGS - good to detect SNP’s in a region of DNA that has already been sequenced so you already have the ‘normal sequence code’
- faster
- cheaper
- shorter reads (50-500bp’s long)
- less accurate
- needs robotics and computers
- parallel sequencing
What are genome wide association studies (GWAS)?
- large population studies (100, 000’s of participants)
- used to work out if people with the same disease have common features on DNA e.g. mutations/SNP’s
- 2 groups are compared (those with and those without the condition)
- the two groups must be well balanced apart from the characteristic being investigated e.g. same ethnicity, race etc. to avoid unimportant SNP’s being identified
- is a measure of ASSOCIATION and not cause (e.g. SNP may highlight a near by mutated gene)
PAPER: JC (Ward et al 2017) (carried out GWAS to find genetic loci associated with mood instability = 4 genomic loci found that were associated with mood instability on chromosomes 8, 9, 14, 18 - clinical use of these results was a bit pointless, and finding a loci does not tell you the involved/responsible gene. Also results were stat. insig. when stratified for age and sex)
What are SNP’s?
Discrepancies in the nucleotide sequence of our DNA = single nucleotide polymorphism
- ~3 million are present in our genome, and are present in > 1% of the population
- Usually found in non-coding regions
- Usually don’t change amino acid sequence
- Can act as genetic markers for disease (e.g. the C282Y mutation in the HFE gene - are associated with hereditary haemochromatosis)
- do not usually represent causality, just associations
What is genetic drift?
- the changes seen overtime in the genotype of a population
- the slow accumulation of genetic changes over time due to the fact that certain genes will be lost overtime if individuals die or fail to reproduce
- occurs due to random sampling of populations
= variation in the frequency of genotypes in a population due to the chance that particular genes disappear as individuals die/do not reproduce.
What is epigenetics?
- heritable changes in a person’s genomic profile as a result of modified gene expression and altered transcription rather than a change in the underlying DNA itself
- mechanisms can be environmental:
= Diet
= PAPER Dolinoy et al 2008 Viable yellow agouti mouse, mother mice fed with high bisphenol A (BPA) diets had offspring with yellow coats, and low BPA black coats
= Smoking
= Carcinogens
= Sleep
= Physical activity - Mechanism which are naturally causing a balance between genetic activation (transcription) and silencing:
-> Histone acetylation
-> DNA methylation
-> MicroRNA’s
Describe genetic differences between individuals and how these can be detected using DNA techniques:
Point mutations, deletions etc. (sequencing) Allelic differences (HLA locus) SNP's (Illumina, GWAS studies) Microsatellite repeats (sequencing) Epigenetic changes Chromosomal amplifications (FISH = fluorescent in-situ hybridisation)
Describe PCR and give an example of its use
Ingredients -> part of genome (DNA) to be sequenced, primer, Taq polymerase enzyme
90o -> DNA denatured
45o -> primers anneal
75o -> Taq polymerase enzyme extends each ssDNA from the position of the primers onwards, making dsDNA
- computer produces a graph by measuring no. cycles against PCR product -> three phases (geometric, linear and plateau) and peak PCR levels are at linear phase (about 30-40 cycles)
Tan et al 2012 -> PCR can be used to check for KRAS point mutations to predict which CRC patients should receive Cetuximab
Describe real time PCR
Ingredients -> part of genome (DNA) to be sequenced, primer, Taq polymerase enzyme and TaqMan probe (which binds to unreplicated DNA single strands)
Probe has a ‘quencher’ molecule attached and a ‘reporter’ molecule attached
Before DNA replicated, the TaqMan probe is intact and the quencher exhibits FRET (fluorescent resonance energy transmission) over the reporter, stopping it emitting fluorescence
- once the probe is no longer intact, the quencher cannot dampen the reporter, and so the reporter emits fluorescence and this is measured by computer
- the intensity of the fluorescence emitted = proportional to the quantity of DNA product
