Techniques Flashcards
Compare the sizes of bacterial and eukaryotic genomes
Bacterial 0.6-10Mb
Eukaryotic 10-100,000 Mb
What are the steps of Sanger sequencing?
Denature DNA
Anneal the primer
Chain extension in presence of a limited number of radioactively labelled dideoxy NTPs (ddNTP)
Incorporation of ddNTP stops DNA strand extension (OH on 3’ of sugar is replaced with H)
E.g. if T is on the template strand, occasionally ddA will be added and chain will terminate. All the products of varying lengths end with an A
Radioactivity can be detected by X-ray film following polyacrylamide gel electrophoresis
Reading sequence from bottom up gives sequence from primer into unknown DNA
Explain modifications to Sanger’s method
Thermal Cycle Sequencing - The original method uses the template once as removing E.coli polymerase kills it - Use of thermostable polymerases meant the reaction could be recycled and reused
Improving Fragment Separation - Using capillary separation means smaller amount of DNA needed, more consistent, faster run times
Fluorescent Detection - Original method meant 4 reactions required for one section of DNA. GE of Taq enzyme allowed ddNTPs labelled with fluorophores means one reaction, detect which light peak produced by each dye as they fall out the tube
Pros and Cons of Sanger sequencing
Pros:
- Cheap per experiment (~£4) - 1 sequence
- Very good single read accuracy (99.9%)
Cons:
- Need to know the first primer
- High cost per million bases (£4000)
What Is The Process Of Shotgun Sequencing?
Break DNA into pieces (enzymatic or physical)
Clone pieces and sequence both ends
Lots of reactions in parallel means lots of fragments that need to be put together and have overlapping sequences that look similar
These are aligned in contiguous sequence (contigs)
Higher number of overlapping sequences means higher chance it is the correct assembly
Bigger pieces are easier to be put together
Interspersed repeats prevent contig expression, Tandem repeats cause contig compression
What is the concept of coverage in relating to DNA sequencing and how accurate is it?
1x coverage = having sequenced DNA of equivalent length to genome size (not all sequenced, some will repeat what’s already sequenced)
Even at 10x coverage, sequence will have many gaps - even 1 small section can make sequence unusable
Explain Illumina Sequencing
Most dominant next gen sequencing method
1. Prepare genomic DNA by smashing it to bits and adding adapters to ends using ligases
2. Attach DNA to surface of glass slide - sparsely bind ss fragments randomly to inside surface of flow cell channels
3. Bridge amplification by PCR reaction - unlabelled nucleotides and enzyme
4. Fragments become ds - bridges on solid phase substrate
5. Denature ds molecules - ss templates anchored to substrate
6. Complete amplification - several dense clusters of dsDNA (all DNA in cluster is the same) generated in each channel of flow cell
7. Add 4 labelled reversible terminators, primers and polymerase, reaction stops due to terminator - wash of unincorporated nucleotide and polymerase
8. Image 1st base by laser excitation, detect fluorophore to see what was incorporated, cleave dye and unblock 3’ OH
9. Sequence over multiple cycles determines bases one at a time
10. Align data and compare to reference
Pros And Cons Of Illumina Sequencing
Cons:
- Not as simple to apply as Sanger
- Set up is expensive (£1-20k per experiment)
- Limit to fragment size (~500bp) – have to be short enough to generate a small cluster and not reach across to other side of the glass chip
- Limit to how many times you can repeat the chemical cycle (up to 300b)
Pros:
- Capable of generations 10s to 1000s of million sequences in parallel
- Cheap cost per million bases (2-20p)
- Very good single base accuracy (99.9%)
Explain Nanopore Sequencing
Individual nanopores embedded in synthetic membrane
Voltage creates current through pore
Molecules passing through affect magnitude of current
Loop adapter joins 3’ end of one strand to 5’ end of other strand
5’ adapter provides binding for enzyme which feeds DNA into pore (slows DNA down)
Ss moves through pore
Different DNA causes different deflection of current
Pros And Cons Of Nanopore Sequencing
Pros:
- Not sequencing by synthesis – don’t have to worry about number of cycles or if polymerase falls offf
- Can process 100s of bases per second
- Can also sequence modifications to DNA/RNA
- Sequencing can continue until nanopore blocks to membrane breaks down
- In theory, can sequence extremely long fragments of DNA
Cons:
- Much noisier than sequencing 1000s of copies at once as nucleotides don’t go through the pore one at a time - approx. 5 bases in pore at a time so (45) 1024 possibilities
- Single read accuracy is much lower than Sanger/Illumina (90% but improving)
What are the methods of measuring mRNA Levels?
Northern Blotting
Quantitative Reverse Transcriptase PCR
RNA Sequencing
Explain Northern Blotting
Separate RNA molecules by size by agarose gel electrophoresis then transfer RNA to supportive membrane
Add labelled ssDNA which hybridises to cmRNA
Wash off unbound DNA
Amount of bound prove proportional to amount of mRNA in sample
Can use labelled probe to see levels of specific transcripts
Complex target, simple probe
Shows size as well as amount
Test level of one transcript at a time
Explain qRT-PCR
Regular PCR repeated denaturation (94C), annealing of primers (55-65C) and DNA synthesis (72C) amplifies in exponential fashion
RT produces cDNA from RNA, RT tends to snap back at end, making hairpin loop
Endpoint amount depends on available resources not necessarily amount of input material
Transforming volume to log scale means you can trace back to original amount. observe at which point DNA amount crosses threshold
Can detect amplified DNA by using DNA-binding dyes (e.g. SYBR Green I) – Unbound dye low fluorescence; bound dye high fluorescence- fluorescence increases as more dsDNA is produced – works with any amplicon but can’t distinguish between real product and mis-priming
Explain RNA-Seq
With NGS, alternative is to sequence all cDNA; under ideal conditions, number of sequences arising from each cDNA will be proportional to the level of mRNA
Allows analysis of all mRNAs (transcripts) at once – transcriptomics (not measuring transcription) – lots of power – much more expensive than single-gene/mRNA measurement
Why are there difficulties in measuring protein levels?
Proteins cannot be generically isolated from cells based on chemical characteristics
Generally aren’t linear in natural state so won’t run neatly in gel, don’t have consistent charge
Can’t use binding of complementary sequences to detect
Explain Western Blotting
Separate proteins by size by SDS-PAGE (sodium dodecyl sulfate) which has negative charge and binds to protein evenly, keeping it linear
SDS overpowers any natural charge
Use charge to send through polyacrylamide gel mesh
Transfer denatured protein to supporting membrane e.g. nitrocellulose
Block non-specific sites on membrane
Incubate with primary antibody and wash
Incubate with secondary antibody (with marker e.g. fluorophore) and wash and detect by horse radish peroxidase by chemiluminescence
Reagent = H2O2 and luminol
HRP catalyses breakdown of H2O2 to H2O and O2
Luminol is oxidised and emits photon of light, detected on film/camera
How can protein tagging be used as an alternative to making a specific antibody?
Fuse CDS for protein of interest to a genetically-encoded tag e.g. GFP
Can follow as it goes along the cell
Recombinant Protein Production - what does a generic plasmid look like?
Coding Sequence (CDS) for protein
DNA encoding protein of interest often dropped onto MCS
Machinery around the gene
DNA to enable replication of plasmid (ori - specific to bacteria)
Need to be able to select for bacteria that have taken up the plasmid e.g. resistance to drug most common
Using lac operon
Contains genes for lactose metabolism, operator (lacO), regulatory gene for repressor (LacI)
Lactose binds to repressor protein which falls off operator and allows operon to be transcribed
Can use to make toxic product combining machinery from lac operon and polymerase from bacteriophage
Isopropyl beta-D1-thiogalactopyranoside = non-metabolisable analog of lactose
When IPTG is present in hose cell, it causes lac repressor dissociation and allows T7 RNA polymerase to initiate target gene transcription
