Molecular Techniques Flashcards
What is cloning?
taking a gene from its native source and moving it to a new vector (such as plasmid).
Where do restriction enzymes originate?
Part of the restriction/modification system of prokaryotic organisms
What type of restriction enzyme is most often used?
That which produces sticky ends and has high specificity to one place in the DNA
Describe restriction enzyme/ligation cloning.
- cleave DNA of interest with restriction enzyme
- Cleave cloning vector with the same restriction enzyme
- remove cut part of plasmid with electrophoresis
- recreate closed, circular dsDNA by linking the cut parts of DNA using ligase (ATP-dependent)
- transform bacteria and use positive selection encoded in plasmid to select for ligated molecules
Describe cloning vector pUC57
- oriC
- amp resistance
- cloning region with many restriction sites
- B-galactosidase for color screening (white colonies are cloned, blue are not)
What are the natural processes by which foreign DNA gets into bacteria cells?
Transformation = DNA in surrounding environment
Conjugation = sex pillus
Transduction = bacteriophage
How can transformation be promoted in E. coli?
It is not normally competent, so we must pre-treat with calcium or electroporation.
What are two common bacteria cloning strains?
- DH5a
- TOP10
these are mutated to lessen homologous recombination and endonuclease activity.
What is a common protein expression bacteria strain?
BL21(DE3):
- lacking proteases
- optimizes T7 polymerase/lac operon systems
- can incorporate chaperones, disulfide isomerases, rare tRNAs
Describe the E. coli expression vector.
- oriC
- promotor
- affinity tags
- coding sequence for tag removal
- multi cloning sequence (restriction sites)
- inserted gene sequence
- selection marker (can be cut out later)
What is the purpose of making a genome library?
To find a gene sequencing
Why are genome libraries not often used anymore?
Because we do DNA sequencing and PCR more often
Describe how to make a genome library.
- randomly shear DNA with 4-bp cutters for a partial digest (will cut once every 256 base pairs to generate DNA fragments in uniform length)
- fragments are then inserted into vectors
Describe the vector types used to make a genome library.
- plasmid: limited to 10,000 bp of DNA
- phage: can package larger pieces of DNA into phage (30,000bp)
- BAC: bacterial artificial chromosome, can hold 100,000bp. hard to purify because it is present in only one copy in the bacteria
- YAC: yeast artificial chromosome, can hold several million bp.
Why do we make cDNA?
It can give us a look into what RNA is expressed at a certain time by making a more stable nucleic acid
How is cDNA made and cloned?
- oligoT primer hybridizes to the poly A tail of mRNA
- reverse transcriptase synthesizes DNA, using mRNA as a template, in 5’->3’ direction
- RNA is degraded with RNAseH, leaving behind a small RNA primer to use in synthesis of second DNA strand
- second strand of DNA is synthesized by DNA polymerase
Describe the PCR reaction.
- heat to separate strands (denaturation)
- cool to anneal primer pair (annealing)
- synthesize DNA with temperature stable polymerase (elongation)
How many reps of PCR are performed?
About 30. Each replicate doubles what we have (2^n)
Describe the drawbacks of Taq polymerase
- error prone and lacks 3’->5’ proofreading activity
- can only do short products
we therefore will compensate with other polymerases
What are the benefits of using vent polymerase?
has 3’->5’ proofreading exonuclease
-has high thermostability: long half-life
What is the benefit of using long-range polymerase?
-can amplify up to 30k (solution to short products by Taq)
-
How does one select for a good primer pair?
- melting temp between 50-65 (two primers need same Tm, and we reanneal at 4 degrees below this)
- absence of dimerization capability between the pair
- absence of hairpin formation within a single primer
- lack of secondary priming sites
- primer length (18-22bp)
How can PCR be used to diagnose duchennes muscular distrophy?
you can amplify exon 46. In those with the disease, when you amplify with PCR and run on a gel there will be no band when you probe for the exon.
How can PCR be used to add sequences?
- Because the 3’ end of the primer needs to be stable but the 5’ end does not, we can get an overhang at the ends of the DNA sequence we want to add to.
- the overhang will then be filled in by DNA polymerase, and the new ends are made (perhaps with new restriction sites or a tag)
When would we use PCR to add sequences?
If we want to clone a piece of DNA into a vector but it does not have the restriction sites we need to do so.
How can we tell if a yeast gene is successfully knocked out using PCR?
We can use a positive selection marker to replace the gene being deleted. PCR can be used after digestion to look for difference in restriction digest bands.
What is a positive yeast selectable marker?
kanomycin
What is another name for direct PCR cloning?
Ligation independent cloning
Describe ligation free cloning
- cut cloning vector with restriction enzyme
- prepare insert with several base homology (~15bp) to the vector ends using PCR.
- mix insert with linearized vector
- treat with T4 DNA polymerase to chew back dsDNA (3’-5’ exonuclease activity) and leave 3’ overhangs that anneal together
- then put in E. coli to ligate, because it has its own ligase
What are the cons to ligase-free cloning?
efficiency of cloning is only 10-25%
What are the benefits to ligase-free cloning?
- high throughput, so can screen for many transformed colonies at once using PCR
- can quickly and cheaply clone same insert into many types of vectors and is cheap!
- can clone many insert boundaries into the same vector which is good for screening functional regions of a gene
How is In-Fusion enzyme used?
it works like T4 DNA polymerase to do ligase-free cloning of multiple gene fragments all in one tube
What is assembly PCR good for?
Production of synthetic genes and even synthetic genomes.
How does assembly PCR work?
- oligonucleotides with overlapping regions are extended in PCR
- primers at the end of the DNA molecule allow for amplification of the target sequence
What is codon optimization?
switching the codons used in a transgene without changing the amino acid sequence that it encodes for in order to replace rare codons with those matching more common tRNAs. Increases the abundance of the protein expression dramatically
What is allele-specific PCR?
It is a diagnostic or cloning technique based on single nucleotide variations. A specific primer can only amplify a normal or mutant allele, but not both within the same reaction.
What is nested PCR used for?
It reduces non-specific binding in PCR products due to the amplification of unesxpected primer binding sites.
How does nested PCR work?
- Two sets of primer pairs are utilized
- the first round of PCR may have nonspecific primer binding elsewhere than the target sequence resulting in an unwanted product
- second round uses different primers that won’t hybridize to the first unwanted product, but will hybridize within, or nested in, the first, wanted product.
What is degenerate PCR?
Uses degenerate PCR with primers that are degenerate in their 3rd base to amplify DNA sequences that are unknown but related to a known sequence. Degenerate primers are a mix of primers that differ in the last position.
What is RT-PCR useful for?
- detecting transcripts of really any gene
- eliminates need for abundant starting material for northern blot
- provides tolerance for RNA degradation as long as RNA sequence spanning the primer is in tact.
How is gene expression measured using RT-qPCR?
- Incorporate sybrgreen into dsDNA as it is replicated. More fluorescence from sybr green indicates more DNA. the earlier the fluorescent midpoint (i.e. less cycles), the more DNA there is.
- OR, Taqman probe bound to DNA being amplified. as it is amplified, the probe is displaced and cleaved, causing it to emit fluorescence. Measured and interpreted the same way as sybrgreen
Describe Sanger DNA sequencing.
- anneal primer to DNA sequence
- DNA polymerase extends the primer using regular nucleotides and ddNTPs that are 32P labelled (not many of these), and uses these at random
- the ddNTPs when incorporated terminate the elongation.
- then run gel. Each lane is a different reaction using A,T,C or G. The furthest band to run is the earliest in the sequence. (end of gel = 5’, beginning of gel = 3’)
Describe automated dideoxy sequencing.
- done like PCR, but only one primer set so that amplification is not exponential
- uses ddNTPS with each a different color fluorophore so the reaction can happen all in one tube.
What is a tilling array?
It is a whole genome microarray, but the probe design is different. The tilling probes are from known contiguous sequences. Resolution power depends on probe design, i.e. how much they are spaced apart or are overlapping.
What is the general outline of high throughput sequencing?
- obtain DNA or RNA (requires conversion to cDNA) from single cell or population of cells
- PCR
- DNA is fragmented and ligated to adaptors specific for to the sequencing procedure. (adaptors are needed for clustering)
- sequencing is performed using “sequence by synthesis” (a variation of Sanger sequencing)
Describe clonal single molecule array
Uses adaptors to fill a chip with amplified DNA strands
