The document discusses various vector systems used for molecular cloning, including plasmids like pBR322 and pUC, phages such as λ phage and cosmids, and phagemids. λ phage and cosmids can accept large DNA inserts up to 20-50kb, making them useful for genomic libraries. M13 phages produce single-stranded recombinant DNA useful for sequencing and mutagenesis, while plasmids called phagemids also generate single-stranded DNA with helper phages.

1. Molecular Cloning Methods

2. Vectors serve as carriers to allow
replication of recombinant DNAs.
 Origin of replication
 Multiple cloning site(MCS)
 Selection gene
Plasmids pBR322 pUC
Phages λphage cosmids M13
Phagemids

4. Summary:Summary:
The first generations of plasmid cloning vectors were pBR322 andThe first generations of plasmid cloning vectors were pBR322 and
the pUC plasmids. The former has two antibiotic resistance genes and athe pUC plasmids. The former has two antibiotic resistance genes and a
variety of unique restriction sites into which one can introduce foreignvariety of unique restriction sites into which one can introduce foreign
DNA. Most of these sites interrupt one of the antibiotic resistanceDNA. Most of these sites interrupt one of the antibiotic resistance
genes, making screening straightforward. Screening is even easier withgenes, making screening straightforward. Screening is even easier with
the pUC plasmids. These have an ampicillin resistance gene and athe pUC plasmids. These have an ampicillin resistance gene and a
multiple cloning site that interrupts a partial β-galactosidase gene. Onemultiple cloning site that interrupts a partial β-galactosidase gene. One
screens for ampicillin-resistant clones that do not make active β-screens for ampicillin-resistant clones that do not make active β-
galactosidase and therefore do not turn the indicator, X-gal, blue. Thegalactosidase and therefore do not turn the indicator, X-gal, blue. The
multiple cloning site also makes it convenient to carry out directionalmultiple cloning site also makes it convenient to carry out directional
cloning into two different restriction sites.cloning into two different restriction sites.

5. Figure 4.3 The plasmid pBR322, showing the locations of 11 unique restrictionFigure 4.3 The plasmid pBR322, showing the locations of 11 unique restriction
sites that can be used to insert foreign DNAsites that can be used to insert foreign DNA
The locations of the two antibiotic resistance genes (Ampr =ampicillin resistance;The locations of the two antibiotic resistance genes (Ampr =ampicillin resistance;
Tetr =tetracycline resistance) and the origin of replication (ori ) are also shown.Tetr =tetracycline resistance) and the origin of replication (ori ) are also shown.
Numbers refer to kilobase pairs (kb) from theNumbers refer to kilobase pairs (kb) from the EcoEcoRI site.RI site.

7. Figure 4.4 Cloning foreignFigure 4.4 Cloning foreign
DNA using theDNA using the PstPstI site ofI site of
pBR322.pBR322.
We cut both the plasmid andWe cut both the plasmid and
the insert (yellow) withthe insert (yellow) with PstPstI, thenI, then
join them through these stickyjoin them through these sticky
ends with DNA ligase. Next, weends with DNA ligase. Next, we
transform bacteria with thetransform bacteria with the
recombinant DNA and screen forrecombinant DNA and screen for
tetracycline-resistant, ampicillin-tetracycline-resistant, ampicillin-
sensitive cells. The recombinantsensitive cells. The recombinant
plasmid no longer confersplasmid no longer confers
ampicillin resistance because theampicillin resistance because the
foreign DNA interrupts thatforeign DNA interrupts that
resistance gene (blue).resistance gene (blue).

8. Figure 4.5 Screening bacteria byFigure 4.5 Screening bacteria by
replica plating.replica plating.
(a)(a) The replica plating process. WeThe replica plating process. We
touch a velvet-covered circular tool totouch a velvet-covered circular tool to
the surface of the first dish containingthe surface of the first dish containing
colonies of bacteria. Cells from each ofcolonies of bacteria. Cells from each of
these colonies stick to the velvet andthese colonies stick to the velvet and
can be transferred to the replica plate incan be transferred to the replica plate in
the same positions relative to eachthe same positions relative to each
other.other. (b)(b) Screening for inserts in theScreening for inserts in the
pBR322 ampicillin resistance gene bypBR322 ampicillin resistance gene by
replica plating. The original platereplica plating. The original plate
contains tetracycline, so all coloniescontains tetracycline, so all colonies
containing pBR322 will grow. Thecontaining pBR322 will grow. The
replica plate contains ampicillin, soreplica plate contains ampicillin, so
colonies bearing pBR322 with inserts incolonies bearing pBR322 with inserts in
the ampicillin resistance gene will notthe ampicillin resistance gene will not
grow (these colonies are depicted bygrow (these colonies are depicted by
dotted circles). The correspondingdotted circles). The corresponding
colonies from the original plate cancolonies from the original plate can
then be picked.then be picked.

10. pUCpUC

11. lacZ’ : coding for the amino
terminalportion of the enzyme β –
galactosidease.
Host E.coli strain carry a gene fragment
that codes the carboxyl potion of β –
galactosidease;
When X-gal cleaved by β –galactosidease, it
releases galactose plus an indigo dye that
stains the bacterial colony blue.

12. Figure 4.7 Joining of vector to insert. (a)Figure 4.7 Joining of vector to insert. (a)
Mechanism of DNA ligase.Mechanism of DNA ligase.
Step 1Step 1:: DNA ligase reacts with an AMPDNA ligase reacts with an AMP
donor—either ATP or NAD(nicotinamidedonor—either ATP or NAD(nicotinamide
adenine dinucleotide), depending on theadenine dinucleotide), depending on the
type of ligase. This produces an activatedtype of ligase. This produces an activated
enzyme (ligase-AMP).enzyme (ligase-AMP). Step 2Step 2:: TheThe
activated enzyme donates a phosphate to theactivated enzyme donates a phosphate to the
free 5’-phosphate at the nick in the lowerfree 5’-phosphate at the nick in the lower
strand of the DNA duplex, creating a high-strand of the DNA duplex, creating a high-
energy diphosphate group on one side of theenergy diphosphate group on one side of the
nick.nick. Step 3Step 3: With energy provided by: With energy provided by
cleavage of the diphosphate, a newcleavage of the diphosphate, a new
phosphodiester bond is created, sealing thephosphodiester bond is created, sealing the
nick in the DNA. This reaction can alsonick in the DNA. This reaction can also
occur in both DNA strands at once, so twooccur in both DNA strands at once, so two
independent DNAs can be joined togetherindependent DNAs can be joined together
by DNA ligase.by DNA ligase.

13. Figure 4.7 Joining of vector to insert.Figure 4.7 Joining of vector to insert.
(b)(b)Alkaline phosphatase prevents vectorAlkaline phosphatase prevents vector
re-ligation.re-ligation.
Step 1Step 1:: We cut the vector(blue, top left)We cut the vector(blue, top left)
withwith BamBamHI. This produces sticky endsHI. This produces sticky ends
with 5’-phosphates(red).with 5’-phosphates(red). Step 2Step 2:: WeWe
remove the phosphates with alkalineremove the phosphates with alkaline
phosphatase, making it impossible for thephosphatase, making it impossible for the
vector to re-ligate with itself.vector to re-ligate with itself. Step 3Step 3:: WeWe
also cut the insert(yellow, upper right)also cut the insert(yellow, upper right)
withwith BamBamHI, producing sticky ends withHI, producing sticky ends with
phosphates that we do not remove.phosphates that we do not remove. StepStep
44:: Finally, we ligate the vector and insertFinally, we ligate the vector and insert
together. The phosphates on the inserttogether. The phosphates on the insert
allow two phosphodiester bonds toallow two phosphodiester bonds to
form(red), but leave two unformedform(red), but leave two unformed
bonds, or nicks, These will be completedbonds, or nicks, These will be completed
once the DNA is in the transformedonce the DNA is in the transformed
bacterial cell.bacterial cell.

14. Phages as vectors
Natural advantages over plasmid:
They infect cells much more efficiently
than plasmids transform cells, so the yield
of clones with phage vectors is usually
higher.

15. Summary:Summary:
Two kinds of phages have been especially popular as cloningTwo kinds of phages have been especially popular as cloning
vectors. The first of these is λ, from which certain nonessential genesvectors. The first of these is λ, from which certain nonessential genes
have been removed to make room for inserts. Some of these engineeredhave been removed to make room for inserts. Some of these engineered
phages can accommodate inserts up to 20 kb, which makes them usefulphages can accommodate inserts up to 20 kb, which makes them useful
for building genomic libraries, in which it is important to have largefor building genomic libraries, in which it is important to have large
pieces of genomic DNA in each clone. Cosmids can accept even largerpieces of genomic DNA in each clone. Cosmids can accept even larger
inserts—up to 50 kb—making them a favorite choice for genomicinserts—up to 50 kb—making them a favorite choice for genomic
libraries. The second major class of phage vector is composed of thelibraries. The second major class of phage vector is composed of the
M13 phages. These vector have the convenience of a multiple cloningM13 phages. These vector have the convenience of a multiple cloning
site and the further advantage of producing single-strandedsite and the further advantage of producing single-stranded
recombinant DNA, which can be used for DNA sequencing and forrecombinant DNA, which can be used for DNA sequencing and for
site-direct mutagenesis. Plasmids called phagemids have also beensite-direct mutagenesis. Plasmids called phagemids have also been
engineered to produce single-stranded DNA in the presence of helperengineered to produce single-stranded DNA in the presence of helper
phages.phages.

16. Figure 4.8 Cloning in Charon 4.Figure 4.8 Cloning in Charon 4.
(a)(a) Forming the recombinant DNA.Forming the recombinant DNA.
We cut the vector (yellow) withWe cut the vector (yellow) with EcoEcoRIRI
to remove the stuffer fragment and saveto remove the stuffer fragment and save
the arms. Next, we ligate partiallythe arms. Next, we ligate partially
digested insert DNA (red) to the arms.digested insert DNA (red) to the arms.
(b)(b) Packaging and cloning thePackaging and cloning the
recombinant DNA. We mix therecombinant DNA. We mix the
recombinant DNA from (a) with anrecombinant DNA from (a) with an inin
vitrovitro packaging extract that contains λpackaging extract that contains λ
phage head and tail components and allphage head and tail components and all
other factors needed to package theother factors needed to package the
recombinant DNA into functionalrecombinant DNA into functional
phage particles. Finally, we plate thesephage particles. Finally, we plate these
particles onparticles on E.coliE.coli and collect theand collect the
plaques that form.plaques that form.

17. Figure 4.9 Selection of positiveFigure 4.9 Selection of positive
genomic clones by plaquegenomic clones by plaque
hybridization.hybridization.
First, we touch a nitrocellulose otFirst, we touch a nitrocellulose ot
similar filter to the surface of the dishsimilar filter to the surface of the dish
containing the Charon 4 plaques fromcontaining the Charon 4 plaques from
Figure 4.8. Phage DNA releasedFigure 4.8. Phage DNA released
naturally from each plaque will sticknaturally from each plaque will stick
to the filter. Next, we denature theto the filter. Next, we denature the
DNA with alkali and hybridize theDNA with alkali and hybridize the
filter to a labeled probe for the genefilter to a labeled probe for the gene
we are studying, then use X-ray filmwe are studying, then use X-ray film
to reveal the position of the label.to reveal the position of the label.
Cloned DNA from one plaque nearCloned DNA from one plaque near
the center of the filter has hybridized,the center of the filter has hybridized,
as shown by the dark spot on theas shown by the dark spot on the
film.film.

18. Cosmids
Behave both as plasmids and as phages;
Contain the cos sites of λ and plasmid origin
of replication;
Have room for 40-50 kb inserts.

19. M13 phage vectors
β –galactosidease gene fragment
pUC family MCS
Single stranded DNA genome

20. Figure 4.10 Obtaining single-Figure 4.10 Obtaining single-
stranded DNA by cloning in M13stranded DNA by cloning in M13
phage.phage.
Foreign DNA (red), cut withForeign DNA (red), cut with HinHindIII, isdIII, is
inserted into theinserted into the HinHindIII site of the double-dIII site of the double-
stranded phage DNA. The resultingstranded phage DNA. The resulting
recombinant DNA is used to transformrecombinant DNA is used to transform
E.coliE.coli cells, whereupon the DNA replicatescells, whereupon the DNA replicates
by a rolling circle mechanism, producingby a rolling circle mechanism, producing
many single-stranded product DNAs. Themany single-stranded product DNAs. The
product DNAs are called positive (+)product DNAs are called positive (+)
strands, by convention. The template DNAstrands, by convention. The template DNA
is therefore the negative (-) strand.is therefore the negative (-) strand.

21. PhagemidesPhagemides
Single-stranded;Single-stranded;
Both phage and plasmid characteristics;Both phage and plasmid characteristics;
Help phageHelp phage
Two RNA polymerase promoters (T7andTwo RNA polymerase promoters (T7and
T3)T3)

23. Summary
Two kinds of phages have been especially popular as cloning vectors. TheTwo kinds of phages have been especially popular as cloning vectors. The
first of these is λ, from which certain nonessential genes have been removedfirst of these is λ, from which certain nonessential genes have been removed
to make room for inserts. Some of these engineered phages can accommodateto make room for inserts. Some of these engineered phages can accommodate
inserts up to 20 kb, which makes them useful for building genomic libraries,inserts up to 20 kb, which makes them useful for building genomic libraries,
in which it is important to have large pieces of genomic DNA in each clone.in which it is important to have large pieces of genomic DNA in each clone.
Cosmids can accept even larger inserts—up to 50 kb—making them aCosmids can accept even larger inserts—up to 50 kb—making them a
favorite choice for genomic libraries. The second major class of phage vectorfavorite choice for genomic libraries. The second major class of phage vector
is composed of the M13 phages. These vector have the convenience of ais composed of the M13 phages. These vector have the convenience of a
multiple cloning site and the further advantage of producing single-strandedmultiple cloning site and the further advantage of producing single-stranded
recombinant DNA, which can be used for DNA sequencing and for site-recombinant DNA, which can be used for DNA sequencing and for site-
direct mutagenesis. Plasmids called phagemids have also been engineered todirect mutagenesis. Plasmids called phagemids have also been engineered to
produce single-stranded DNA in the presence of helper phages.produce single-stranded DNA in the presence of helper phages.