The Genetic Revolution Flashcards
Explain Sanger Sequencing
Dideoxynucleotides (ddNTPs), as opposed to deoxynucleotides, prevent furthur DNA synthesis. **Missing a hydroxyl group.
- 4 reactions set up, each contains:
- Single stranded template (that you want to know the sequence of)
- Small amount of radioactive tracer
- Specific primer
- One of the 4 dideoxy-NTPs
- dNTPs
- DNA synthesis takes place but ocassionally the DNA polymerase will add a ddNTP, terminating synthesis
- Run the 4 reactions side by side on a gel to read sequence. (fragments will be different sizes so will run to different spaces on the gel)
What modification have their been to sanger sequencing to increase throughput?
- Using flourecently labelled primers, allowing the 4 reactions to be pooled after synthesis and detected by a laser
- Using flourecently labelled ddNTPs so that reaction could be performed in a single tube `
Describe illumina sequencing
- Single DNA molecules are bound to the surface of a flow cell and each molecule is amplified by PCR to give a cluster of copies
- Sequencing begins, but only florescent, “reversible-terminator” dNTPs are present
- In each cycle, only one florescent dNTP can be added, as this terminated synthesis. A excites that fluorophores on the fragments and and image of the cell is taken.
- The dye on the dNTPs is removed, so that one more dNTP can be added and another image taken
- This is repeated again and again
Pros and cons of illumina sequencing
Very accuracte
Relativley short reads at once (150-300bp)
Describe nanopore sequencing
- Nucleic acids are captured and fed though a pore by a motor protein
- As they pass through the pore, there is a change in electrical current, which is different for each of the 4 nucleotide bases, which can be recorded
Pros and cons of nanopore sequencing
Extremley long reads (100-300kb)
Is more error prone than otehr techniques
- BUT this is new (10year old) technology so improvements are happening rapidly
How may a genome be sequenced by clone-based approach?
(top down)
BACs: bacterial artificial chromosome, engineered cloning vector
1. Large set of clones (such as BACs) are ordered in an overlapping way to span the whole genome
2. Select the smallest set of clones that span the whole genome
3. Individual BACs are furthur broken down and sequenced
3. Overlapping areas must be identified to place sequenced DNA in correct order
- A very laborious and slow technique
Describe shotgun sequencing as a way to read genomes?
- Entire genome is randomly fragmented and each small section of DNA is sequenced
- Computer algorithms are used to compare all sequences and assemble them into a continuous sequence
How can DNA microarrays be used to measure gene expression?
-
DNA microarrays: solid supports where DNA fragments can be placed in an ordered array eg. could have fragments of many different genes
- 2 different RNA samples are labelled with different coloured dyes, mixed together, and hybridised to same microarray (probably doesnt necessarily have to be 2 samples)
- Array can then be scanned by lased and determine the intensity of the florescent signal (a measure of ‘how strongly’ gene is expressed
Describe an alternative approch to microarrays and hybridisation to determine RNA levels
mRNA-seq
1. Remove DNA and rRNA from sample
2. Use reverse transcripase to convert mRNA into cDNA
3. Fragment the DNA and read.
- can be used to identify isoforms (formed from alternative splicing)
Much more sensitive than microarrays and has mostly replaced them
Why is RNA sequencing so important?
- Can be used to determine timing/conditions that specific genes are expressed and therefore can be correlated to their fuction in the body or in development
- Can see which genes are being upregulated/downregulated in cancer cells and therefore the genes that could be potencial drug targets for treatment
What is the use of ChIP-seq?
Chromatin Immunopurification
Used to identify where along the genome TFs (or other proteins) associate with DNA
Describe how ChIP-seq works
- Formaldehyde (or other chemical crosslinking agent) is used to ‘stick’ TF and DNA together
- DNA is then isolated from the sample and fragmented (200-500bp)
- Antibody that recognises the protein of interest is added. Therefore, will also seperate out any DNA bound to that protein
- Crosslinks reversed and any DNA extracted can be purified and read to identify where the protein was bound (after PCR)
Describe SNP detection by microarrays
- Short set of probes that can discriminate between a known SNP (eg. a T or a G)
- Arrays containing SNP probes are hybridsed with genomic DNA from individuals and can determine if they are homozygous for one allele or heterozygous
Descibe 2D PAGE
2-Dimensional Polyacrylamide Gel
Electrophoresis. The original proteonomic method
- Proteins separated on the basis of charge and then in a second dimension based on molecular mass
- Can run two gels in parallel and compare to identify differences in protonome
- Could also cut individual spots from teh gel and analyse to identify that specific protein
Cons: labour intensive, low throughput, can only detect <10% of proteonome
