DNA analysis and DNA sequencing Flashcards
what can an empty plasmid do but not a selectable marker
-the empty plasmid can also transform host cells – selectable marker cannot do this for you.
-Your cloning strategy should minimise this
-Still need to screen transformants – restriction enzymes / PCR
-Simplest scenario:
=Empty vector
=Incorrect orientation
=Other weird stuff (occasionally: no plasmid, truncations)
-Often sequence to check for mutations – but need to know before this, whether it’s the right thing – screen several transformants
what prep is needed when getting the DNA out of bacteria
-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 (hopefully recombinant) DNA
How do we analyse the size of restriction fragments / PCR products
-Electrophoresis:
=Here of DNA molecules. But can be used for RNA and proteins too
=Here, separation is by size (variations can use other characteristics of molecules, such as charge)
what are The techniques that we used for analysing results of molecular cloning
-We can predict DNA sizes from RE digest or PCR and see if we get the predicted sizes or something else.
-If we’re clever about choice of REs / design of primers, we can distinguish between multiple possibilities (confirm correct cloning)
How do we know what DNA we have when analysing it
-Samples loaded into the wells
-Electric current causes migration of samples. DNA is negative so it moves towards the positive electrode
-Small fragments move more quickly; large fragments more slowly
-Dye to visualise DNA (Ethidium Bromide / Midori Green). Intercalates into DNA and fluoresces under UV light
-DNA ladder to estimate size
whats the cloning strategy
-Cloned insert using a single RE site (=1)
-But insert has other RE sites in it (= enzyme 2)
-RE 2 and 3 will show different patterns
-1 distinguishes between +/- insert (but not orientation)
-2+3 will allow you to assess the orientation
-Different-sized DNA fragments can be distinguished by gel electrophoresis
-Sometimes necessary to do more than one diagnostic digest
analysis of the cloning strategy
-Cloned insert using a single RE site (=1)
-Design some primers specific to vector and insert
-Helps to add primers to products
- Will only get a PCR product with correct cloning product
-Sometimes presence/absence of a PCR product can be our diagnostic test (must have appropriate controls – here would have some other controls)
-Sometimes the size is what we are looking for
-Can also combine PCR with RE digest
how can PCR be used for diagnosing
-Modern genetic fingerprinting
-Identification of repeat expansions (certain diseases)
-Identification of genomic rearrangements (cancer
how can restriction digestion be used in analysing
-Similar with RFLP – number of repeats affects the size of the restriction fragment
-This technique requires a way to detect specific DNA fragment – Southern blot
-Or a mutation can destroy or create a RE site
-It might be necessary to combine this with PCR of a specific DNA fragment
how can you check your DNA sequence
-Cloning: check there are no PCR-induced mutations
-Sequence a gene (WT versus mutant)
what does sequencing rely on us being able to work out
-How long a DNA molecule is (how many base pairs)
-What the last nucleotide is
-Then we can know the identity of a nucleotide at a specific position
how does DNA sequencing work
-Specialised form of DNA synthesis
-DNA is amplified meaning lots of fragments
-For each fragment:
=We know what the last nucleotide is
=We can measure DNA size
-Bit like PCR, but only one primer
-Cycles of synthesis means lots of pieces of DNA (all same template strand)
-For each amplified fragment, we always know what the last nucleotide is (PCR includes a chain terminator)
-And we can measure its size
what are modified dNTPs
-Difference at 3’ position – no OH group
-Can’t form a phosphodiester bond with the 5’ phosphate of the next nucleotide meaning termination of sequencing
-Inclusion of only one ddNTP means we know what the last nucleotide is (ddATP / ddCTP / ddTTP / ddGTP)
-Let’s see an example
-dideoxynucleoside triphosphate (ddNTP)
-deoxynucleoside triphosphate (dNTP)
what is the Basis of DNA sequencing
-The chain terminator stops DNA synthesis specifically at the nucleotide “A”
-All DNA fragments will have A as the last nt (we don’t know anything else about them right now)
-We can use electrophoresis to work out how big these fragments are
-look at slide
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.
what originally was automated sequencing
-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
whats the new modern automated sequencing
-One reaction contains all four ddNTPs – with 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
what are the Components of a sequencing reaction
-A DNA polymerase
-Deoxynucleotides (dNTPs)
-Dideoxynucleotides (ddNTPs)– fluorescently labelled- ddATP, ddCTP, ddGTP, ddTTPBuffer (including Magnesium)
-Template DNA – plasmid / PCR product
-Primer - We usually use a primer that binds the vector, Can design more primers once we know the sequence
