DNA sequencing Flashcards
Molecular cloning
Potential problems:
- Empty vector
- Incorrect orientation
- Occasionally no plasmid, truncations
Sequence to check for mutations
Screen several transformants
First get DNA out of bacteria
Mini-prep:
- Grow lots of bacteria (several ml of culture)
- Break them open
- Plasmids are small and compact
- Genomic DNA is big and tangled and precipitates with other stuff (proteins, lipids, etc.)
- Results in µg of purified plasmid DNA
Colony might contain millions of bacteria but need lots more
Then use molecular biology techniques to combine
Same techniques as used for cloning
Need to be smart about restriction enzymes and design primers so can distinguish between multiple copies and confirm the correct clone
Restriction digest:
- Digest DNA with a specific restriction enzyme
- Look whether you get right sized DNA fragments
PCR:
- Use DNA as template in PCR
- Then work out if got right sized PCR product
How to tell what DNA you have
Electrophoresis
Technique used to separate DNA or RNA or protein fragments based on size
Two types: DNA electrophoresis or agarose gel electrophoresis
How to do it:
- Load samples into wells
- Electric current causes migration samples, DNA negative so moves towards positive electrode
- Small fragments move quicker and large move slower
- Visualise using dye ~ initially ethidium bromide but safer alternatives exist now
Cloning strategy and restriction mapping
Clone insert using single restriction enzyme site (RE 1)
But insert has other restriction enzyme site (RE2)
Using multiple restriction enzymes will allow you to ensure that they are cutting at the correct site or if there is incorrect orientation or an empty vector
RE1 distinguishes between a +/- insert but not orientation
RE2 and 3 allows you at assess orientation
Use g Electrophoresis to distinguish between different sized DNA fragments
Sometimes will need to do more than one diagnostic digest
DNA analysis using PCR - cloning strategy
Cloned insert using single RE site
Design primers specific to vector and insert
Helps add primers to products, pay particular attention to orientation
Will only get PCR product with correct cloning product
Sometimes presence/absence PCR product can be diagnostic test
Sometimes size is what were looking for
Can also recombine PCR with RE digest
If all good will have PCR product with a pair of primers working
If its an empty vector there is no PCR product then only one primer worked
if its incorrect orientation then no PCR product and primers both amplify in the same direction and not opposite directions
PCR - Not just for cloning
Modern genetic fingerprinting - Genetic fingerprinting amplifies regions that contain repeats (number of repeats is specific to each individual)
Identification of repeat expansions (certain diseases) - Huntington’s (e.g.) – mutant allele has more repeats
Identification of genomic rearrangements (cancer)
Both can be diagnosed by PCR
size of PCR product is different
bit of an oversimplification
other tests also exist (RFLP)
Restriction digest - Not just for cloning
Restriction Fragment Length Polymorphism
Similar with RFLP – number of repeats affects the size of the restriction fragment
Or a mutation can destroy or create a RE site
(This can be combined with PCR of a specific DNA fragment or some other detection method – more on those next time)
DNA sequencing
The ultimate way to check your DNA sequence
Cloning: check there are no PCR-induced mutations
Sequence a gene (WT versus mutant)
Sequencing is a specialised form of DNA synthesis
If there is some way that we can stop DNA synthesis at a known nucleotide…
And we know what size that DNA molecule is…
Then we can work out what nucleotide is at a specific position
Sequencing is much like PCR – in vitro DNA synthesis
If we can stop synthesis at A and we know that piece of DNA is 10 nt long we know the nt at position 10 is an A
Basis of DNA sequencing
Need to know:
What the last nucleotide is
How long the DNA molecule is
if we stop DNA synthesis specifically at the nucleotide “A” we have a variety of fragments that all have A as the last nt (we don’t know anything else about them right now)
Can use electrophoresis to work out how big fragments are and where they are like position 1 5 and 7 and then do some of the other bases
Modified dNTPs
deoxynucleoside triphosphate - dNTP (has Oh at 3’ carbon)
dideoxynucleoside triphosphate - ddNTP (has H at 3’ carbon)
ddNTP can’t for phosphodiester bond with 5’ phosphate at next nucleotide - terminates sequence
Problems to solve
How do we know how long the DNA molecule is? Gel electrophoresis – discriminate between DNA molecules by size
How do we know which base is last? Separate reactions with different ddNTPs (ddATP, ddTTP, ddGTP, ddCTP)
If sequencing terminates at the first A (e.g.) how will we ever know the rest of the sequence? We add both ddNTP and “normal” dNTPs. Millions of products. Each will incorporate the ddNTP (and hence terminate) in a different place.
Each sequencing reaction produces millions (more!) of DNA synthesis products. Some of one size, some of another
DNA sequences contain multiple A nucleotides
If sequencing terminates at the first A, we will never know the rest of the sequence
We don’t want sequencing to stop of the first A – how do we get fragments of different lengths?
Have both dNTPs and ddNTPs (usually about 10:1)
When ddATP incorporated, synthesis stops and that’s what we use to tell then use gel electrophoresis to run all the different sequences and then you can work out what the sequence is
CAn do this with ddTTP, ddGTP, ddCTP to work out with T, G and C
How originally did automated sequences
Four separate reactions, each with a different ddNTP
Reaction used a radioactively labelled primer
Agarose electrophoresis of separate reactions – read by hand
T7 DNA polymerase
Needed a lot of template DNA
Labour-intensive
How its done now
One reaction
Contains all ddNTPs – have different fluorescent labels
Automated capillary gel electrophoresis – read by a computer
Taq DNA polymerase
More sensitive and more specific
Easy, quick and cheap!
One reaction contains all ddNTPs – have different fluorescent labels
Capillary gel electrophoresis – read by a computer
Taq DNA polymerase – PCR-like reaction
More sensitive and more specific
Easy, quick and cheap!
