Topic 3 - Molecular cloning Flashcards
what is a cloning vector?
a relatively small piece of DNA that
- can be added to an organism and maintained
- can insert a foreign DNA fragment into
what is a restriction enzyme? AKA?
protein that cleaves DNA at specific seq (restriction sites)
AKA restriction endonucleases
what is DNA ligase
enzyme that joins DNA together
catalyzes the formation of phosphodiester bonds
features common to ALL cloning vectors (4)
- replicate independently (e.g., ori)
- unique cloning sites (polylinker, multiple cloning sites (MCS))
- carry a selectable marker (e.g., antibiotic resistance)
- easy to purify from host cell
why are plasmids less likely to be attacked by host exonucleases than some other vectors?
circular and small
what does ori stand for
origin of replication
what is ori?
- where replication begins
- different types of plasmids with same ori cannot co-exist in one cell
- cell cannot keep track of which plasmid has been replicated
why can different types of plasmids with same ori NOT co-exist in one cell?
one will outcompete the other in the long-term
If cloning more than one plasmid type into the same cell, they can’t have the same ___
ori
what is the multiple cloning site?
what happens when we use them?
what can we do with them?
- up to ~20 unique restriction sites all adjacent to each other
- AKA polylinker
- these sites occur only ONCE in plasmid; when treated with RE, plasmid is linearized not fragmented
- allows choice of restriction enzymes
- can insert multiple fragments into one region, without disrupting rest of the sequence
T/F: To insert a single gene into a plasmid, we typically need to use multiple restriction enzymes.
False - but sometimes, we would use multiple to control insert direction
what is copy number control? why is it important?
- trait associated with each type of plasmid
- can be high or low copy (high = lots of plasmids; vice-versa)
- important when cloning for high expression of inserted sequence
exactly when would we want high versus low copy number control?
high -> production of things, e.g., insulin
low -> production of things toxic to the cell
what is important about vector sequences being known (what can we do with this info)?
- known restriction map
- can design primers for analysis (e.g., PCR)
additional optional features: promoters
initiate transcription of inserted sequences
- direct expression of ORFs to change expression pattern/amt after construct is introduced into host
- direct expression of a reporter gene to monitor activity of promoter, once construct is introduced into host
We don’t NEED a promoter to transform a plasmid into a host cell. But what would be the consequences of not having a promoter?
- the plasmid gene will NOT be expressed
additional optional features: reporter genes
(explain lacZ ex in depth)
express proteins that are easily detectable (e.g., GFP, lacZ)
- adaptable - MCS in coding region of ORF
- The plasmid contains an incomplete lacZ gene (lacZ’). The host organism has the rest of the gene (need both to have working lacZ)
- When introduced into a host cell with other half of the gene : lacZ is functional
- LacZ codes for the enzyme β-galactosidase (cleaves lactose into glucose and galactose)
- Bacterial colonies expressing lacZ turn blue when grown on media with X-gal (cleaved into galactose and blue compound)
what is blue-white screening? AKA?
Functional LacZ (no insert) = blue
Non-functional LacZ (has insert) = white
AKA alpha complementation
advantages of sticky ends over blunt ends
- easier to attach insert (even without ligase, some can attach temporarily w H bonds)
- nothing holds blunt end insert and plasmid together
when would we want to use blunt ends?
if plasmid and insert do not have complementary ends or matching recognition sites
Overview of cloning process (steps)
step 1: obtain foreign DNA to insert into plasmids
- can be a specific DNA fragment
- can be an entire genome or collection of mRNA -> cDNA
step 2: cut both plasmid and foreign DNA w/ 1 or more REs to make complementary ends
step 3: incubate plasmid and foreign DNA together to let them bind, use DNA ligase
step 4: transform the recombinant DNA molecule into competent host cells
how to prevent plasmid from ligating to itself without insert?
alkaline phosphatase removes 5’ terminal phosphate from each terminal 5’ end of plasmid
- a single strand nick is created, but will not affect transformation and replication
transformation def
uptake of naked DNA by a bacterial host (i.e., not in injection by viral capsid)
cells that can take up DNA through transformation are considered _____
competent
how can we create competent cells? (2 methods)
- method 1: CaCl2 treatment followed by heat shock
- method 2: electroporation
Making competent cells:
CaCl2 treatment + heat shock steps
- grow cells to log (exponential) phase
- collect cells by centrifugation (cells in pellet)
- wash & resuspend in ice-cold CaCl2 (+ve charge neutralizes -ve charge on cell wall, easier for bacteria to uptake naked DNA)
- add DNA (after ligation or intact plasmid as a control)
- leave on ice 20 mins then heat shock at 42C for one min (makes pores in membrane)
- add LB, grow for 1hr at 37C (let cells transcribe inserted gene)
- plate on selective media
chemically competent = ?
- cells made competent using CaCl2 + heat
- lower transformation efficiency than electroporated cells
chemically competent cells usu. have a transformation efficiency of _____________
~10^7 transformants per ug of DNA
Making competent cells:
electroporation steps
- grow cells to log phase
- collected by centrifugation
- wash several times and resuspend in water and glycerol
- add DNA and cells to a special cuvette
- zap with short voltage pulse (millisecs)
- add LB, grow for 1h at 37C
- plate on selective media
easier to kill cells than heat shock!
electroporation:
why do we do this:
3. wash several times and resuspend in water and glycerol
- glycerol to maintain cells cool enough during electroporation
- not just water due to osmotic shock
- salt can alter the electroporation process, ST cells are killed
electrocompetent cells =?
cells made competent through electroporation
usual transformation efficiency for electroporation?
10^8 or more transformants per ug of DNA added
(more than CaCl2 + heat)
transformation efficiency unit?
colonies / mg DNA added
what type of DNA should be used to calculate transformation efficiency? why?
purified supercoiled plasmid
- can’t use linear DNA (degraded by bacteria)
- products of a ligation mix is more DNA than the actual amount that persists in host cell
- purified supercoiled plasmid has exact right conc
what are the 2 types of vectors based on bacteriophage lambda?
- cosmids and replacement vectors (phage)
the lambda genome is a linear molecule (~50kb) w/ _______ ends
- _____ _____ when packaged into capsid
12 bp cohesive ends
(annealing produces a circular structure)
- tightly wound when packaged into capsid
what happens to the bacteriophage lambda upon binding to bacterial host cell?
bacteriophage lambda injects its linear genome into host cell
- immediately, cohesive ends (12bp) of genome anneal -> bacterial ligase fixes the “nick”, circularizing it
- when it circularizes, a COS SITE is created
what is the cos site recognized by?
recognized later in life cycle (after injection) by a lambda-encoded endonuclease
bacteriophage lambda is a ____ virus, meaning:
temperate virus! two possible outcomes of a lambda infection:
- lysogenic
- lytic infection -> we want! can insert DNA!
lambda lytic cycle steps
- binds to protein receptor on EC cell wall
- phage inserts linear DNA into cell
- phage genome circularizes
- several rounds of theta replication
- rolling circle replication creates CONCATAMERS
abt concatamers in lambda lytic cycle
head-to-tail joining of copies of genome
- long piece of DNA with multiple cos sites
- concatamers get stuffed into capsids -> more viruses
how to detect lambda lytic cycle
phage an host cells mix together, plated on solid LB agar
- initial infected cells liberate viral particles, which then infect and lyse surround cells
- creates zones of clearing (plaques) on bacterial lawn
Q: Cos sites are critical for…
a) Packaging of lambda DNA.
b) Expression of lambda genes.
c) Successful infection by lambda.
a)!
packaging of viral DNA (general)
- Viral packaging machinery recognizes the cos sites on concatemers, makes a cut in the DNA, creates a cohesive end, stuffs a “headful” DNA into pre-formed capsid
- Only cares about cos site (good for inserts!)
- Normally head is full by the time the next cos site on concatemer is reached
how to make heads and tails separately (lambda particles)? how to assemble them?
- lambda strains with mutation in head gene can only make tails
- lambda strains with mutation in tail gene can only make heads
- when heads and tails are mixed, functional phage SELF-assembles!
By mixing _________ with these extracts (heads and tails), the cos sites on the cosmid are recognized by packaging enzymes → _________________________
ligated DNA
the recombinant molecule is packaged into the capsids, then tail is added.
_____ of wt size of lambda genome for filled capsid is stable
~75-105%
what are replacement vectors
part of normal viral genome is cut out and replaced with insert DNA
- don’t take out genes necessary for infection
what are cosmids
- a larger plasmid with a cos site (for packaging)
- package by in vitro lambda packaging extracts
- inserts need to be 32-45 kb to be packaged (capsid stability)
- circularizes to form a big plasmid following phage infection
what gene do we not need in cosmids, but do need in replacement vectors?
infection genes!
Q: You infect E. coli cells with lambda phage packed with cosmids. After infection, you plate your cells on an agar plate. You should observe…
a) Colonies. - took out viral genome; should not be inducing further infection and killing cells
b) Plaques.
a)!
genomic library
collection of clones that in total contain all fragments of a genome
- never quite complete irl
- telomeres, centromeres, other highly repetitive regions are hard to clone
why are repetitive sequences hard to include in genomic library?
may not have restriction recognition sites, so too big for insert size limit
possible reasons to make a genomic clone library
- isolate a genomic region
- isolate promoter
- gene mapping
- genome sequencing
creating a genomic library steps
- choose vector
- fragment genome into small pieces to fit into vector
- ligate vector and genomic DNA
- transform recombinant vector into host cells
- eval library quality
- store library
Biggest fragments in a genomic digest are usually ~30-40 kb; why don’t we see larger fragments?
DNA shearing (pipetting, bead beating, etc.)
a genomic library separates genomic fragments into _____ _____
individual cells!
what do we want ideally in a genomic library, for its fragments of genome?
each fragment in digest is ideally ligated into a vector, which is represented by a colony at the end
would we generally prefer a complete or partial digest, for restriction digest for genomic fragments
we would want a partial digest (not every recog site in DNA is cut)
-> will produce overlapping fragments to see how the fragments may link together
restriction digest: what factors create a partial digest?
- short digestion times
- suboptimal Mg conc (common)
- low amount of enzyme
- low temp (less common)
genomic library: how do we ensure there is only one insert per vector?
- we treat the inserts with alkaline phosphatase, NOT the plasmids
- keep insert to vector ratio low, like 3:1
genomic library: would we want to use CaCl2 + heat OR electroporation?
electroporation, because higher transformation efficiency - want to get as many as possible because inserts are all diff
genomic library: how do we evaluate the library?
plate a small amount of transformation mix on selective medium, count number of colonies w/ insert
- need avg insert size & total # of clones w/ vector + insert
- can accomplish by extracting plasmids from a representative number of colonies, looking for a plasmid (w/ gel electrophroesis)
- can also use alpha complementation and count white colonies
evaluating genomic library: without blue-white screening. what do we do?
pick representative cells, determine avg insert size, determine if plasmid is inside (if DNA is on gel or not)
- can treat plasmids with restriction enzyme and remove fragments
- would have plasmid fragment + different insert sizes after restriction enzymes
how should we store libraries?
- -80C long-term
- need cryoprotective agent (e.g., glycerol or DMSO) to prevent dmg from ice crystal formation
- stock never thawed, some of stock is sampled to inoculate medium
amplification of libraries (general)
- take sample of transformation mix or frozen library, inoculate it in fresh media (bacterial library) or fresh cells (phage library)
- cells replicate -> amplify
why might amplified libraries have skewed abundances of certain fragments?
random chance!
what is it called when some genes are always “on” (transcribed)?
constitutively expressed
how are cDNA libraries different from genomic libraries?
instead of genomic DNA, they contain total cellular mRNA seq (in cDNA)
limitations of cDNA libraries
- reflects tissue used to isolate mRNA + its conditions at time of sampling
- difficult to recover long transcripts
- unspliced transcripts are usu not recovered
- difficult to recover rare transcripts; v abundant mRNA will be represented a lot
- mRNAs without poly A tail are usually not recovered
similarities between cDNA and genomic libraries during the making process (after we have cDNA)
- inserting (c)DNA into vector, transform into competent cells
- storage
do we treat mRNA with restriction enzymes to make cDNA
NO! they’re also already short, but now they have blunt ends
how do we purify mRNA from total RNA (mRNA, tRNA, rRNA, etc.)?
- bind RNA to silica membrane under high salt conditions
- affinity chromatography
- column contains a matrix attached to oligo-dT or oligo-dU
– complementary to poly A tail! - wash w high salt
- then elute in low salt buffer like always
after purifying mRNA, how do we make cDNA?
using reverse transcriptase (isolated from retroviruses)
cDNA =?
copy or complementary DNA
how does reverse transcriptase work? what is it?
- RNA-dependent DNA polymerase (template)
- our primer should be a short DNA fragment that’s complementary to 3’ poly A tail (poly (dT))
how do we go from mRNA : cDNA hybrid to ds cDNA? (2 methods)
method 1:
- digest RNA
- add DNA pol 1 (Klenow fragment) + dNTPs
- add S1 nuclease to cleave hairpin
method 2:
- make nicks in RNA with RNase H
- use the RNAs as a primer for DNA synthesis (first cDNA strand serves as template)
technical problems associated with RT
- secondary structures in the template
- cause RT to slow down or fall off, reducing recovery of some sequences
(part of why long transcripts are usu not recovered)
why are more processive and temp-resistant RTs useful?
processive - tends to hold on for longer
- temp can denature secondary structures (more linear RNA to work with)
what vector should we usu use for cDNA library?
plasmids!
cloning cDNA:
after RT: blunt end or 1-nucleotide overhand
how can we get these DNA fragments into plasmids with no restriction sites??
- If our enzyme produces a 1 base overhang, we can make a plasmid with a complementary 1 base overhang
- Add recognition sites to the ends of PCR products so they can be cloned more readily
- Extend the ends of the inserts with terminal transferase (oligo dC) and extend ends of vector with oligo dG
cDNA cloning: adding restriction sites to primers (PCR)
- recognition sites added to 5’ ends of primers so they are copied into synthesized DNA
- restriction enzymes do not cut efficiently close to DNA ends, so extraction nucleotides are added to primer ends
– poly A tail & enzyme restriction site on other site -> repetitive seq on both ends
– additional nucleotides at end of primer
cDNA cloning: with terminal transferase
- adds base pairs without template (control addition of bases by controlling avail nucleotides)
- terminal transferase catalyzes addition of deoxynucleotides to 3’ hydroxyl of DNA
