Recombinant DNA and molecular cloning

Question 1

molecular cloning purification?

Answer 1

only one plasmid will properly integrate into a cell
introducing a mixtire of plasmids - only one particular plasmid would be linked to a bacteria
can clonally expand the plasmid

Question 2

molecular cloning uses?

Answer 2

purifying and amplifying certain genes/DNAs of interest

obtaining DNA sequences (sequencing strategies use cloned vectors)

determining gene structure and regulation

site directed mutagenesis - investigating gene function

express/purify protein fo biochem/structural analysis

enable genome analysis by creating overlapping clones of genomic DNA

reintroduce genes into another organism (transgenesis) to acquire new functions and phenotypic changes

Question 3

how do type II restriction endonucleases work?

Answer 3

recognise short DNA sequences (4,5,6,8 nt) and cut at specific position to produce discrete fragments
only require Mg2+, no atp requirement

most commonly bind palindromic sequences (bind as a 2 subunit HOMODIMER) at cut at a specific nucleotide on each side

bind DNA non-specifically first
then slide along molecule
change conformation to specifically bind and cut after recognising their sequence

some type IIS REs cut outside their recognition site

Question 4

type of type II restriction enzyme cut sites?

Answer 4

3’ staggered/recessed ends - where the 3’ OH end is further back than the 5’ phosphate end
-protruding phosphate

5’ staggered ends other way around
-protruding hydroxyl

blunt ends - no overhangs

Question 5

type II restriction methylases?

Answer 5

restriction enzyme cleavage blocked by methylation added by their cognate methylase

protect bacteria from cleavage by their own phage DNA protection system

both full methylation and hemimethylation block cleavage

e.g. EcoRI methylase blocks cleavage by EcoRI by methylating the EcoRI recognition site

Question 6

DNA ligases?

Answer 6

catalyse formation of 5’-3’ phosphodiester bonds

require a cofactor (rATP for T4 DNA ligase) that forms covalent intermediate with the enzyme

repair dna nicks, join adjacent Okazaki fragments in replication

Question 7

ligase uses in recombinant techniques?

Answer 7

DNA “sticky end” overhangs can associate with each other by H-bonding between complementary bases
stabilises the ends next to each other
DNA ligase can then resetore the phosphodiester bond

REQUIRES the free hydroxyl at 3’ and the phosphate at the 5’ next to it

can also join restiction fragments with blunt ends (i guess complementarity of bases doesn’t matter here??)
reduced efficiency though as no base pairing to stabilise

Question 8

concentration and ligation outcome?

Answer 8

ligase links two restriction fragments with matching ends
e.g. a vector and DNA insert

high concentration of substrate:
intermolecular ligation (end of one molecule joins to end of another)
creates linear CONCATENATED dna
many molecules joined end to end

low concentration:
allows intramolecular ligation
one end of molecule joins to its other
forming covalently closed circular DNA

low concentration allows one end of the DNA insert to join to one end of the plasmid vector (intermolecular)
and then both ends of the joint molecule to join and form circular DNA product

Question 9

DNA polymerase properties?

Answer 9

add new dNTPs to the 3’ hydroxyl of a primer using a template
and so synthesise in 5’-3’ direction

many DNA pol also have 3’-5’ exonuclease proofreading activity to eliminate errors
e.g. E. coli DNA pol I
also 5’-3’ exonuclease activity - removing primers

can use a Klenow sub fragment of DNA pol I
lacking 5’-3’ exonuclease
doesn’t remove primers
so useful for keeping radiolabelled primers in molecule

Question 10

radiolabelled nucleotide properties?

Answer 10

dNTP
has alpha, beta, gamma phosphates (triphosphate)
beta and gamma lost when incorporated into molecule (alpha forms phospodiester bond)
alpha phosphate labelled with 32P (radioactive)

DNA pol used to add this radioactive nucleotide to end of molecule (end labelling)

Question 11

radioactive probe production?

Answer 11

klenow sub fragment used

-denature restriction fragment
-anneal primers (hexanucleotide, randomly chosen so randomly hybridise)
-synthesise labelled DNA strand using alpha-32P dCTP)

-klenow sub fragment so primers aren’t removed - syntheised DNA stays as small fragments

-these fragments can be used as radiolabelled probes against the sequence in that fragment

Question 12

DNA polynucleotide kinase?

Answer 12

T4 poly nt kinase
catalyses transfer and exchange of phosphate from gamma position of ATP to the 5’ phosphate terminus of double and single stranded DNA

used to efficiently label the 5’ end of restriction fragments or synthetic ss oligonucleotides to use as probes
using gamma-32P-ATP

or used to phosphorylate PCR producta made w/out phosphorylated primers so they can be ligated

Question 13

molecular cloning strategies?

Answer 13

vectors are:
-plasmid derived - vector DNA introduced by transfection
-phage derived - vector DNA introduced by transduction (phage infection)
-combination of both - vector introduced by transduction

Question 14

cloning vector properties?

Answer 14

-a selectible genetic marker - constitutively expressed gene encoding antibiotic resistance

-recpilcation origin (ori) for autonomous replicaiton in bacteria

-unique restriction enzyme sites where DNA restiction fragments can be inserted

Question 15

molecular cloning process:

Answer 15

-prepare vector by cutting at unique restriction enzyme site - produce linear molecule

-prepare and purify DNA insert by cutting with same restriction enzyme - or an enzyme that produces same type of sticky ends

-mix DNAs at low conc. and add DNA ligase
products of this will be mix of religated vector and vector with insert (AND unligated DNAs)

-Trasnform into E. coli strain eith optimal genetic features for molecular cloning

Question 16

E. coli transformation process?

Answer 16

can use either:
-CaCl2 streated E. coli + heat shock
-high voltage electroporation

then select by antibiotic resistance
only E. coli that have recevied a plasmid (with or without insert) will survive and produce a colony

Question 17

how to prevent self ligation of vector ends to prevent vectors w/ no insert?

Answer 17

Phosphatase
remove the phosphates from the 5’ ends
now all ends of vector have only hydroxyl termini
ligase cannot form phosphodiester bonds between these

DNA insert still has phosphate at 5’ ends
can still form phosphodiester bond with 3’ ends of vector
but the OH at 5’ ends of vector means that it cannot form bond with 3’ ends of insert but just the one strand on each side is stable enough to keep vector together
other strand between 5’OH of vector and 3’ OH of insert joined in vivo by the E. coli DNA repair system

Question 18

how to tell if insert is in a plasmid that e .coli has received?

Answer 18

pBluscript
-LacZ reporter on vector
-DNA insert site is inside LacZ coding region

E. coli has:
-Plasmid with no insert:
will have beta-galactosidase activity
BLUE

-plasmid with no insert:
-LacZ product disrupted
-no beta-galactosidase activity
has PLASMID + INSERT

blue/white colony test:

E. coli strain expresses inactive version of beta-galactosidase - no working alpha peptide
uninterrupted alpha peptide gene on uninserted vector = E .coli colony will be blue = no insert

interrupted by insert
no functional alpha peptide
white colony

Question 19

screen recombinant plasmids by nucleic acid hybridisation

Answer 19

go through process of making vector
transform E. coli
plate on selective media

then transfer onto nylon filter and denature the DNA

can then hybridise radioactive probes for the insert
wash and then expose to X-ray

positive result from autoradiograph will correspond to colonies on plate with vector + insert

Question 20

Blunt end cloning

Answer 20

can ligate ends with different restriction cut sites by making them blunt
though blunt ends not as efficient (no base pair stabilisation)

-use DNA polymerase (klenow pol I, or T4 DNA pol) to extend recessed 3’ ends into blunt ends

T4 DNA pol 3’-5’ exonuclease activity used to make blunt ends from 5’ recessed ends
(digest away extended 3’ ends because you cant elongate from 5’ end)

Question 21

linker ligation?

Answer 21

can add a specific restriction end onto a blunt ended molecule
use linker:
-duplex oligonucleotide with recognition sequence of restriction endonuclease in it

add many linkers to end of DNA by blunt end ligation (can do this at high conc to increase efficience)
can then digest with that specific restriction endonuclease
leaves sticky end for that enzyme on the end on the old blunt ends

Question 22

adapter ligation?

Answer 22

adapter is small oligonucleotide
has blunt end with normal 5’ and 3’ ends

also has sticky end for specific endonuclease site with a modified 5’ to have an OH terminus

only blunt end can be ligated to the molecule as sticky end has no phosphate

after ligation to the blunt ended molecule you want to adapt
can use polynucleotide kinase and ATP to restore the phosphates to the 5’ end of the sticky ends

Question 23

Type I restriction modification system?

Answer 23

bind to their recognition site on the DNA
but then cut randomly
therefore are no good for cloning
but if system is present in E. coli strain - could potentially destroy any foreign non-methylated DNA that happens to have a recognition site (including the vector)

and so need to use genetic variants of E .coli w/out this system (k12 strain)
hsdR- strain has type I restriction enzyme R deleted - no plasmid degradation

Question 24

traits of E. coli cloning strain:

Answer 24

No type I REs

also:
insert DNA can be rearranged or deleted resulting from homologous recombination

need to use strain with mutations in the RecA gene (RecA protein helps sequence find homologous sequences in HR)
prevent spontaneous rearrangement between repeated sequences

RecA mutants also more sensitive to UV (less effivient DNA repair as no HR) - increases biosafety as easier to kill with UV sanitation

most K12 strains also contain mutation in endA gene encoding DNA specific endonuclease - improves quality of DNA isolated from the strain

Question 25

PCR for cloning/amplifying?

Answer 25

PCR obtains pure DNA fragments in tube

requires prior knowledge of target sequence
3’ end of PCR primers must be complementary to target sequence over 17-25 nt

synthesised product of copying reaction is used as template in next cycle
exponential amplification from repeated reaction cycles
results in massively amplified population os presominantly identical dsDNA molecules (an amplicon)
length of them represents distance of the primer pair

Question 26

products of each PCR cycle

Answer 26

1st:
primers oriented with 3’ end pointing in direction to be synthesised
first synthesis will go past sequence where other primer anneals
end up with products that are much longer than distance between primers of variable length

2nd: DNA denatured again
renatured to allow primer annealing
primer is able to anneal to product of first cyle
this time extension from teh end of the primer has to stop at the sequence where the other primer binds - as it is the 5’ end of the template
this strand corresponds to distance of primers

3rd (exponential stage):
products of second cycle now can be used
produce duplex DNA products that correspond to distance between primers
exponential amplification of this as the template produces defined predominant size product
more likely for this shortest one to be synthesised as smaller fragments are preferentially syntheised in PCR

longer variable fragemnts are minor product - in very low conc so won’t show up on gel

Question 27

adding nucleotide sequence onto amplicon ends by PCR?:

Answer 27

only 3’ end of primer needs complementarity
can have extra nucleotides on 5’ end
these extra nucleotides will be incorporated into amplicon products
now contain sequence in the 5’ end of the primer

can be used to add appropriate endonuclease restriction sites onto end of PCR product
then cut with enxyme to give appropriate sticky ends

Question 28

issues with Taq polymerase?

Answer 28

no proofreading
but need to use it as it is needed to be used at ~95C (thermostabe)
no proofreading = short replication range as mistakes are more likely to be made
mistakes cause Taq pol to no longer be able to continue

mistakes don’t matter so much for analytical purposes

but for cloning can’t have these mistakes
can use blend of Taq pol and a proofreading pol
decreases mistake rate and increases range of how long a sequence can be replicated

Question 29

bacteriophage lambda properties

Answer 29

linear DNA in phage head
circularised via cohesive/sticky ends after introduction into cell
region containing cohesive ends = cos site

lysogenic pathway:
DNA on circularising is integrated into site in E. coli chromosome
no deleterious effect here
replicated along with bacterial genome
while in this state, cells can be induced to become non-lysogenic and enter lytic pathway

lytic pathway:
happens after relaease of DNA from head
circularises
undergoes theta replication:
-rolls out concatenates of linear genome from rolling replication of circular genome
-Ter enzyme then cuts concatenate at cos sites to produce unit length phage genomes
-genomes packaged into new phages produced by expression of other phage genes
-causes cell death but lots of phages produced

Question 30

lambda packaging in vitro?

Answer 30

use packaging mixtures:
-have two lysogenic E. coli
-each lysogenic strain contains inactive phage DNA missing packagning components that the other contains
-when lysates of these cells are mixed the packaging mechanisms now can be active
-extracts combined with concatenated lambda DNA - extracts will package this into phage heads
-infectious phages formed

-can use this to introduce recombinant lambda phage DNA into phages in vitro

Question 31

lambda as a cloning vector?

Answer 31

size limited
lambda genome cannot support large inserts
inserts can be just a little more than a couple kb

however there are parts of lambda genome not needed for lytic pathway
remove them and make space for more insert DNA

lambda insertion vector generation:
-cleave out non-essential, ligate genome back together
small deletion in non-essential region allows for insertion of up to 10kb
insertions don’t have to be at deletion site

lambda replacement vector generation:
-non -essential region removed and replaced by “stuffer region” containing restriction sites - can be used to insert
-this stuffer region ensures that phage genome is BIG enough to be packaged (too small = also bad)
-inserts can be up to 23kb

lambda DNA + insert ligated at high conc to form concatenates
concatenate genome with inserts can be added to packaging mixture
where Ter cuts at cos sites and genomes with inserts are packaged into phages

no insert = phage genome to small to be packaged
-built in selection against genomes with no insert being packaged

Question 32

cosmid vectors?

Answer 32

vector containing both plasmid and phage components
A plasmid containing a lambda cos site and a restriction site for cloning

cut with RE
ligate at high conc.with inserts to form concatenates
cut with Ter at cos site in packaging mix
individual cosmids then incorporated into phage heads

cosmid = 15kb
above normal average unit length size packagning mix will cut and package

also allows creation of vectors with larger inserts than even a standard plasmid

useful for creating genomic libraries

Question 33

cloning particular gene from complex genome?

Answer 33

-constructing a genomic or cDNA library that contains representation of all DNA/RNA sequences present in the starting material followed by screening the library to identify the desired clone

-use PCR directly to amplify specific sequences from genomic DNA or cDNA (cDNA made from reverse transcription of mRNA)

Question 34

genomic DNA library construction?

Answer 34

cloning of overlapping DNA fragments generated by partial digestion of genomic DNA with a restriction enzyme (that cuts multiple times giving many different endpoints)
-creates multiple segment endpoints within population of fragments
- so that in final library for any point in the genome there will be points from individual clones that overlap

-enzyme used to make partial digest and degree of partial digestion depends on the choice of vector insert size capacity

Question 35

why partial digestion for making genome library?

Answer 35

complete digestion makes too small of fragments (many nearby sites)
aren’t compatible with insertion due to small size

instead partial digestion - followed by size selection gives overlapping fragments
(many cells so many copies of genome - partial digestion so different sites cut on different fragments - hence overlap)

Question 36

genomic library from lambda replacement vector?

Answer 36

stuffer removed
2 arms ligated to restriction fragment
DNA ligated and packaged
-genome with no insert - not packaged
-genome with stuffer - packaged, but stuffer region contains negative selevtion genes that inhibit phage lytic growth (red/gam genes)

lambda library plaques from transfected E. coli being plated transferred onto nylon filter at high density
screened by hybridisation with radioactive probes for gene of interest
OR
are picked into 96 well plates for PCR screening with primers for gene of interest

positive plaques are used to infect more fresh cells to amplify that phage with gene of interest even more

Question 37

genome library with cosmid vector

Answer 37

total cell DNA partial digestion
size fractioning of fragments
~35kb fragments - compatible with cosmid vector (cut at restriction site)
-ligated at high conc to make concatenate with cosmid vector
-selection for insert (see earlier)
-colonies instead of plaques

-each colony represents one clone containing the same insert
-different colonies have different inserts (probably)
-each colony can be picked and screened for gene of interest

Question 38

Chromosome walking? (genomic libraries)

Answer 38

-several clones detected with same probe (so are overlapping)
-terminal regions of these clones can be used to identify new probes that will detect other overlapping inserts in other clones
-overlapping clones constitute a “contig”
-can repeat this to “walk” along the genome and probe outwards to identify all the clones
-

Question 39

genomic library with BAC vector?

Answer 39

BAC = engineered derivative of E. coli F factors, replicate at 1-2 copies per cell (due to Ori) with stable maintenance of cloned inserts

advantage - takes much larger inserts at 250-300kb
larger the clone fragment - less clones needed for complete library coverage
BAC reasonably high efficiency in electroporation into cell

BAC carries antibiotic resistance
colonies robotically picked into wells

DNAs prepared and isolated for identification with PCR probes for gene of interest

Question 40

complementary DNA (cDNA) library properties?

Answer 40

cDNA from reverse transcription of cell’s mRNA
representative of the mRNA population in the cell type from which they were derived

small numbers of different mRNAs are represented at high numbers
While a large number of other mRNAs are represented at very low levels

if mRNA of interest is low level one - need lots of screening to recover it

Question 41

cDNA production?:

Answer 41

extract eukaryotic mRNA via its PolyA tail (polyT complementary - fish it out with that)

reverse transcriptase used to make DNA copy of mRNA
get rid of mRNA
then put a primer at 5’ end without polyA tail - can synthesise second strand with DNA pol

Question 42

cDNA integration into vector?

Answer 42

methylate cDNA at restriction sites for linker you are adding (so they dont get cut)
add restrivtion enzyme sites onto blunt ends with linkers
cut with restriction enzyme

now have ds-cDNA
compatible for cloning into vectors as an insert (like earlier)

Question 43

screening methods in cDNA library?

Answer 43

hybridisation by DNA /RNA radiolabelled oligonucleotide probes

by pcr using specific primers

using Ab to expressed proteins

functional cDNA screens in appropriate cell types

Question 44

cloning by PCR?

Answer 44

if even only partial sequence knowledge:
can use pool of degenerate oligonucleotide primers that contain every possible sequence combo for e.g. a certain sequence of amino acids in the gene product

these primers will amplify up sequence of interest

then clone into vector

Question 45

applications of sequences from molecular cloning?

Answer 45

reporter expression vectors - cloning transcriptional regulation sequence of interest

shuttle vectors w/ separate replication origins, selection markers and promoters for use in prokaryotic and eukaryotic cells, for functional cDNA screening

Question 46

components of a reporter plasmid?

Answer 46

cloned cDNA - could contain transcriptional regulatory region, or a specific UTR of a gene of interest
IRES allowing initiation of translation in mRNA from vector
reporter (GFP, luciferase, LacZ…)

fluorescent markers allow fluorescent activated cell sorting

Question 47

reasons for site directed mutagenesis of cloned DNA?

Answer 47

• change AA sequence
  -create/remove RE site
  -alter gene regulatory regions to define key sequences

Question 48

site directed mutagenesis of cloned DNA method?:

Answer 48

have two oligonucleotide primers (different directions) with the desired mutation on them

then denature parent strands

anneal primers

replication

now have 2 ds circular DNA molecules (heteroduplex - one parent strand and one new strand with mutation in it)

now denature and reanneal
get products:
parent strands together
parent strand with either of the new mutated strands
both mutated strands

the parent strands have mehtylation at GATC sequence (propertie of E. coli strand)
Dpnl cuts here
Dpnl unusual - only cuts at methylated sites
new strands not methylated
Dpnl destroys any of the original DNA - including new strands attached
no now only product of just new mutated strands remain

Question 49

dna construct assembly strategies?

Answer 49

Golden gate assembly
type IIS REs create unique cohesive ends allowing efficient simultaneous assembly of multiple components by ligation in vitro
no restriction sites remain at ligation joining points at final assembly

Gibson assembly
allows DNA molecules to be joined by annealing of short end regions of complementarity in in vitro reaction
can enable multi-segment assemblies up to 100s kb
no “scar” at joins

Question 50

golden gate assembly ?

Answer 50

Type IIs REs cleave away from their recognition site
results in staggered ends as one strand cleaved 1bp away while other cleaves 5 away (for Bsa I)

so can position BsaI sites flanking sequences that need to be joined on each DNA molecule

then cut and ligate (can do in same tube)

Question 51

adding Bsa I sites via PCR?:

Answer 51

can include BsaI sites in teh 5’ end of oligonucleotide primers for PCR

then use vector digested by BsaI
then can digest fragments with Bsa I
ligate everything together

assembled DNA product vector with sequences joined

Question 52

gibson assembly?:

Answer 52

one step
assemble multiple dsDNA fragments
no restriction enxymes
low error rate

two ds DNA molecules with overlap in sequence at the end

use T5 exonuclease which digests away 5’ end (5’-3’ exonuclease)

leaves exposed single stranded region at overlap of molecules

so they anneal together by complementary base pairing

use PFU polymerase to extend the 3’ ends where they reannealed
catches up with T5 exonuclease and stops it
Taq ligase seals nick

2 fragments ligated together in reaction that takes a few minutes with addition of 3 enzymes

this happens at both ends of each fragment so can eventually get circular molecule

can make overlapping region easily with PCR (extra NTs on 5’ end of primer method)

Question 53

how to use gibson assembly to put 2 fragments into a vector?

Answer 53

can clone into vector by creating overlaps at each end of each fragment (using PCR - put overlap into 5’ of primer) AND at the end of teh vector