Chapter 13: Recombinant DNA Technology

Outline:

Molecular Cloning

Overview of Basic

Principals of Cloning

Overview of Vectors

Genomic Libraries

Various virus based

vectors

Molecular Cloning The discovery of restriction endonucleases in the early 1970s led not only to the possibility if

analyzing DNA more effectively but also to the ability to cut different DNA molecules so that

they could later be joined together to create new recombinant DNA fragments. The newly

created DNA molecules heralded a new era in the manipulation, analysis and exploitation of

biological molecules. This process termed gene cloning has enabled numerous discoveries

and insights into gene structure regulation and function. Since their initial use methods for

the production of gene libraries have been steadily refined and developed. Although the

polymerase chain reaction, or PCR, has provided shortcuts to gene analysis there are still

many cases where gene cloning methods are not only useful but are as absolute

requirement.

Recombinant DNA technology depends on the ability to produce large numbers of identical DNA molecules (clones). Clones are typically generated by placing a DNA fragment of interest into a vector DNA molecule, which can replicate in a host cell. When a single vector containing a single DNA fragment is introduced into a host cell, large numbers of this fragment are reproduced along with the vector. Once the genomic DNA is isolated and purified, it is digested with restriction endonucleases. These enzymes are the key to molecular cloning because of the specificity they have for particular DNA sequences. It is important to note that every copy of a given DNA molecule from a specific organism will give the same set of fragments when digested with a specific enzyme. By digesting complex genomic DNA from an organism it is possible to reproducibly divide its genome into large number of small fragments, each approximately the size of a single gene. Some enzymes cut straight across the DNA to give flush ends or blunt ends. Other restriction enzymes make staggered single strand cuts, producing short single stranded projections at each end of the digested DNA. These ends are not identical but complementary and will base pair with each other, they are therefore known as cohesive or sticky ends. In addition the 5’ end projections of the DNA will always retain the phosphate group.

Ligation of DNA molecules The discovery of DNA ligases in 1967 by the Gellert, Lehman, Richardson, and Hurwitz laboratories was a watershed event in molecular biology. By joining 3_-OH and 5_-PO4 termini to form a phosphodiester, DNA ligases are the sine qua non of genome integrity. They are essential for DNA replication and repair in all organisms. Ligases were critical reagents in the development of molecular cloning and many subsequent ramifications of DNA biotechnology, including molecular diagnostics and SOLiD sequencing methods. Ligases are elegant and versatile enzymes and are enjoying a research renaissance in light of discoveries that most organisms have multiple ligases that either function in DNA replication (by joining Okazaki fragments) or are dedicated to particular DNA repair pathways, such as nucleotide excision repair, base excision repair, single-strand break repair, or the repair of double strand breaks via nonhomologous end joining. Genetic deficiencies in human DNA ligases have been associated with clinical syndromes marked by immunodeficiency, radiation sensitivity, and developmental abnormalities The DNA products resulting from restriction digestion to form sticky ends may be joined to any other DNA fragments treated with the same restriction enzyme. Thus, when the two sets of fragments are mixed, base pairing between sticky ends will result in the annealing of fragments that were derived from different starting DNA. There will, of course, also be pairing of fragments derived from same starting DNA molecules, termed re annealing. All these pairings are transient, owing to the weakness of hydrogen boneding between the few bases in the sticky ends, but they can be stabilised by the use of an enzyme, DNA ligase, in a process termed ligation. This enzyme, usually isolated from bacteriophage T4 and called

T4 DNA ligase, forms covalent bond between the 5’-phosphte at the end of one strand and the 3’-hydroxyl of the adjacent strand. The reaction is ATP dependent and often carried out at 10 deg C to lower the kinetic energy of the molecules, so as to reduce the chances of base paired sticky ends parting before they have been stabilized by ligation. However, long reaction times are needed to compensate for the low activity of DNA ligase in the cold. It is also possible to join the blunt ended DNA molecules except that the efficiency of the reaction in much lower in this case. [1] Each DNA molecule is inserted by ligation into the vector DNA molecule, which allows the whole recombined DNA to then be replicated indefinitely within microbial cells. DNA ligases seal 5’-PO4 and 3’-OH polynucleotide ends via three nucleotidyl transfer steps involving ligase-adenylate and DNA-adenylate intermediates.DNAligases are essential guardians of genomic integrity, and ligase dysfunction underlies human genetic disease syndromes. Crystal structures of DNA ligases bound to nucleotide and nucleic acid substrates have illuminated how ligase reaction chemistry is catalyzed, how ligases recognize damaged DNA ends, and how protein domain movements and active-site remodeling are used to choreograph the end-joining pathway. Although a shared feature of DNA ligases is their envelopment of the nicked duplex as a C-shaped protein clamp, they accomplish this feat by using remarkably different accessory structural modules and domain topologies.

Figure 1: DNA Ligation reaction

Figure 2:Three-step pathway of nick sealing by DNA ligase [1]

Figure 3: Ligation of sticky ends: two DNA molecules, cleaved with EcoRI and ligate to form recombinant molecules

Formation of Cohesive ends Cohesive ends permit a linear DNA molecule packaged inside the head of a phage particle to become circular inside the host cell. Thus, the presence of cohesive ends on the DNA molecules of temperate phages fits into the scheme for recombination between a circular phage DNA and the host chromosome leading to insertion of the phage DNA into the chromosome as proposed by Campbell (1962).

Figure 4: Addition and cleavage of a chemically synthesized linker. Producing sticky ends with adaptors.

Plasmid and Basic Cloning Principle

Many bacteria contain an extrachromosomal

element of DNA, a plasmid, which is a relatively

small, covalently closed circular molecule, carrying

genes for antibiotic resistance, conjugation or the

metabolism of unusual substrates. Some plasmids

are replicated at a high rate by bacteria and are

hence excellent potential vectors. In the early

1970s a number of natural plasmids were artificially

modified and constructed as cloning vectors by a

complex series of digestion and ligation reactions.

Figure 5: General features of a Plasmid

One of the most notable plasmids, termed pBR322 after its developers Bolivar and

Rodriguez, was widely adopted. Its main features are:

i) A plasmid is a much smaller than a natural plasmid, which makes it more

resistant to damage by shearing, and increases the efficiency of uptake by

bacteria, a process called transformation.

ii) A bacterial origin of DNA replication ensures that the plasmid will be replicated by

the host cell. Some replication origins display stringent regulation of replication, in

which rounds of replication are initiated at the same frequency as cell division.

Most plasmids, including pBR322 have a relaxed origin of replication, whose

activity is not linked tightly to the cell division, and so plasmid replication will be

initiated far more frequently than chromosomal replication. Hence a large number

of molecules will be produced per cell.

iii) Two genes coding for resistance to antibiotics have been introduced. One of

these allows the selection of cells which contain plasmid: if cells are plated on

medium containing an appropriate antibiotic, only those that contain plasmid will

grow to form colonies. The other resistance gene can be used for the detection of

those plasmids that contain inserted DNA.

iv) There are single recognition sites for a number of restriction enzymes at various

points around the plasmid, which can be used to open or linearize the circular

plasmid. Linear plasmid allows a fragment of DNA to be inserted and the circle

closed. The variety of sites makes it easy to select the enzyme that would be

suitable to the insert and the vector both. The presence of an insert can be

detected by loss of resistance to that antibiotic. This is termed insertional

inactivation.

Figure 6: Multiple Cloning Site (MCS)

Insertional inactivation is a useful selection method for identifying recombinant vectors with

inserts. For example a fragment of chromosomal DNA digested with BamH1 would be

isolated and purified. The plasmid pBR322 would also be digested at a single site, using

BamH1. BamH1 cleaves to give sticky ends, and so it is possible to obtain ligation between

the plasmid and the digested DNA fragments, in the presence of T4 DNA ligase. The

unwanted molecules can be eliminated during subsequent steps. The products of such

reactions are usually identified by agarose gel electrophoresis.

The ligated DNA is then used to transform E.Coli. Bacteria do not normally take up DNA

form their surroundings, but can be induced to do so by prior treatment with Calcium at 4

deg cel., then they are said to be competent, since DNA added to the suspension of

competent cells will be taken up during a brief increase in temperatures termed heat shock.

Small circular molecules are taken up most efficiently, whereas long linear molecules will not

enter the bacteria.

After brief incubation to allow the expression of the antibiotic resistance genes, the cells are

plated onto medium containing an antibiotic, for example ampicillin. Colonies that grow on

these plates must be derived from cells containing plasmid, since this carries the gene for

resistance to ampicillin. To further distinguish the colonies containing the plasmids with

inserts and the empty vectors, the colonies are replica plated using a sterile velvet pad, onto

plates containing tetracycline in their medium. Since BamH1 site lies within the tetracycline

resistance gene, this gene will be inactivated by the presence of the insert, but will be intact

in those plasmids that have merely

recircularized.

Colonies that grow on ampicillin but

not on tetracycline are the ones

containing plasmids with inserts.

Although recircularised plasmid can

be selected against, its presence

decreases the yield of recombinant

plasmid containing inserts. If the

digested plasmid is treated with the

enzyme alkaline phosphatase prior

to ligation, recircularization will be

prevented, since this enzyme

removes

the 5’

phosphate

groups

essential

for ligation.

Links can still be made between the 5’ phosphate of the insert and

the 3’ hydroxyl if the plasmid, so only recombinant plasmids and

chains of linked DNA fragments will be formed. It does not matter

that only one strand of the recombinant DNA is ligated, since the

nick will be repaired by bacteria transformed with these molecules.

First recombinant DNA produced: Stanley Cohen and Herbert

Boyer cut 2 plasmids with EcoRI, ligate and screen for E.coli

Figure 7: Replica plating for detection of recombinant plasmids

Figure 8: First recombinant experiment

clones that are resistant to both antibiotics since they harbor the recombinant plasmids. (Patent 4,237,224, 1980)

The valuable pBR322 have been enhanced by the construction of a series of plasmids

termed pUC (produced by University of California). There is an antibiotic resistance gene for

tetracycline and origin of replication for E. Coli. In addition the most popular restriction sites

are concentrated into a region termed multiple

cloning site (MCS). In addition the MCS is part

of a gene in its own right and codes for a

portion of a polypeptide called beta

galactosidase. When the pUC plasmid has

been used to transform the host cell E.Coli, the

gene may be switched on by adding the

inducer Isopropyl- β-D-thiogalactosidase

(IPTG). Its presence causes the the enzyme β-

galactosidase to be produced. The functional

enzyme is able to hydrolyse a substance

called 5-bromo-4-chloro-3-indoyl galactosidase

(X-Gal) to a blue insoluble material. However if

the gene is disrupted by the insertion of a

foreign fragment of DNA, a non functional

enzyme results that is unable to carry out

hydrolysis of X-Gal. thus a recombinant pUC plasmid may be easily detected, since it s gene

is fully functional and not disrupted. This elegant system termed blue/white selection allows

the initial identification of recombinants to be undertaken very quickly and has been included

in a number of subsequent vector systems.

Figure 10: Schematic of Molecular cloning

Figure 9: Vector map of pBR322

Cloning Vectors For the cloning of any molecule of DNA it is necessary for that DNA to be incorporated into a

cloning vector. These are DNA elements that may be stably maintained and propagated in a

host organism for which the vector has replication functions. A typical host organism is a

bacterium, such as Escherichia coli, that grows and divided rapidly. Any vector with a

replication origin in E.Coi will replicate efficiently. Thus, any DNA cloned into a vector will

enable the amplification of the inserted foreign DNA fragment and also allows any

subsequent analysis to be undertaken. In this way the cloning process resembles the PCR,

although there are some major differences. By cloning, it is possible not only to store a copy

of any particular fragment of DNA but also to produce unlimited amounts of it.

PolyLinkers Chemically synthesized polylinker containing one copy of several different restriction sites is introduced into the vector to facilitate directional cloning.

Figure 11: Polylinker sequence and application

Aspects of Gene Libraries There are two general types of gene libraries: a genomic library, which consists of the total

chromosomal DNA of an organism; and a cDNA library, which represents the mRNA from a

cell or tissue at a specific point in time. The choice of the particular type of gene library

depends on a number of factors, the most important being the final application of any DNA

fragment derived from the library. If the ultimate aim is understanding the control of protein

production for a particular gene or its architecture, then genomic libraries must be used.

However, if the goal is the production of new or modified proteins, or the determination of

tissue specific expression and timing patterns, cDNA libraries are more appropriate. The

main consideration in the construction of genomic or cDNA libraries is therefore the nucleic

acid starting material. Since the genome of an organism is fixed, chromosomal DNA might

be isolated from almost any cell type in order to prepare genomic DNA. In contrast, however,

cDNA libraries represent only the mRNA being produced from a specific cell type at a

particular time in the cells development. Thus it is important to consider carefully the cell or

tissue type from which the mRNA is to be derived in the construction of cDNA libraries.

Genomic DNA Libraries Genomic libraries are constructed by isolating the complete chromosomal DNA from a cell,

and digesting it into fragments of the desired average length with restriction endonucleases.

This can be achieved by partial restriction digestion with an enzyme that recognizes

tetranucleotide sequences. Complete digestion with such an enzyme would produce a large

number of very short fragments, but, if the enzyme is allowed to cleave only a few of its

potential restriction sites before the reaction is stopped. Each DNA molecule will be cut into

relatively large fragments. Average fragment size will depend on the relative concentrations

of DNA and restriction enzyme and in particular, on the conditions and duration of

incubation. It is also possible to produce fragments of DNA by physical shearing, although

the ends of the fragments may need to be repaired to make them flush ended. This is

achieved by using a modified DNA polymerase termed Klenow polymerase. The mixture of

DNA fragments is then ligated with a vector, and subsequently cloned. If enough clones are

produced there is a very high chance that any particular DNA fragment such as a gene will

be present in at least one of the clones. To keep the number of clones to a manageable size,

fragments about 10 kb in length are needed for prokaryotic libraries, but the length must be

increased to about 40 kb for mammalian libraries.

cDNA Libraries There may be several thousand different proteins being produced in a cell at any given time,

all of which have associated mRNA molecules. To identify any one of those mRNA

molecules clones of each individual mRNA have to be synthesized. Libraries that represent

the mRNA in any particular cell or tissue are termed cDNA libraries. mRNA can be used

directly in cloning as it is too unstable. However, it is possible to synthesize complimentary

DNA molecules (cDNAs) to all the mRNAs from the selected tissue. The cDNA may be

inserted into vectors and then cloned. The production of cDNAs is carried out using an

enzyme termed reverse transcriptase, which is isolated from RNA containing retroviruses.

Reverse transcriptase is an RNA dependent DNA polymerase and will synthesize a first

strand DNA complimentary to an mRNA template, using a mixture of four dNTPs. There is

also a requirement for a short oligo nucleotide primer to be present. With eukaryotic mRNA

bearing a poly(A) tail, a complimentary oligo (dT) primer may be used. Such primers provide

a free 3’ hydroxyl group that is used as a starting point for the reverse transcriptase.

Following the synthesis of the first DNA strand, poly(dC) tail is added towards 3’ end, using

terminal transferase and dCTP. This will also, incidentally, put a poly (dC) tail on the poly(A)

of mRNA. Alkaline hydrolysis is then used to remove the RNA strand, leaving single

stranded DNA that can be used, like the mRNA, to direct the synthesis of a complimentary

DNA strand. The second strand synthesis requires oligo (dG) primer, base paired with the

poly(dC) tail, which is catalyzed by the klenow fragment of DNA polymerase 1. The final

product is double stranded DNA, one of the strands being complimentary to the mRNA. One

further method of cDNA synthesis involves the use of RNase H. Here, synthesis of the first

strand cDNA is carried out as above with reverse transcriptase but the resulting mRNA-

cDNA hybrid is retained. RNase H is then used at low concentration to nick the RNA strand.

The resulting nicks expose 3’ hydroxyl groups, which are used by DNA polymerase as a

primer to replace the RNA with a second strand of cDNA.

Figure 12: Genomic and cDNA libraries

Virus based vectors A useful feature of any cloning vector is the amount of DNA it may accept or have inserted

before it becomes unviable. Inserts greater than 5kb increase plasmid size to the point at

which efficient transformation of bacterial cells decreases markedly, and so bacteriohages

(bacterial viruses) have been adapted as vectors in order to propagate larger fragments of

DNA in bacterial cells. Cloning vectors derived from lambda bacteriophages are commonly

used, since they offer an approximately 16 fold advantage in cloning efficiency in

comparison with the most efficient plasmid in cloning vectors.

Lambda phage Vectors Lambda phage is perhaps the most well characterized temperate bacteriophage. Infection by

lambda can result either in lysis (death of the host cell) or in lysogeny (persistence of the

viral prophage in the host cell, with minimal viral gene expression). A small proportion

(~0.1% or less) of lysogenic bacteria undergo spontaneous reactivation of the prophage,

produce viral particles and die. Lysogenic cultures can also be treated in such a way that

most or all of the cells will undergo lysis. This is refered to as induction, and it typically

involves the exposure of bacteria to UV light, X-rays or other DNA damaging agents

(interestingly, these agents also activate the replication of several mammalian viruses,

including HIV-1 and adeno-associated virus).

The regulation of lambda reproduction is very well studied, and it involves a genetic switch that regulates the transition between lysogeny and lysis. In the lysogenic bacterium, only a single phage protein is produced -- the cI repressor protein. This binds to two operators on the viral genome and thereby shuts off the transcription of all of the other genes of the phage. When DNA damage occurs in the bacterial cell, the RecA protein is activated, and

converted into a protease, which degrades cI. This allows for the onset of lytic gene expression. Lambda phage is an important model system for the latent infection of mammalian cells by

retroviruses, and it has also been widely used for cloning purposes. It remains in use for

purposes of cDNA cloning and expression, as well as for genomic DNA cloning.

Figure 13: Life cycles of Lambda Phage vector

Life Cycle of Lambda λ - Lytic Cycle, Lysogenic Cycle The phage DNA on entering the host cell may sometimes immediately multiply and enter the lytic cycle. During this cycle the phage genes are expressed and the phage DNA replicated, leading to the production of many phage particles. This part of the life cycle is virulent, or intemperate. The virus exists in the viron form, with a DNA core in a protein coat (head) and a tail.

In the lysogenic cycle the viral chromosome becomes integrated into the host chromosome and is called a prophage. When the viral DNA becomes a part of the host DNA it behaves like a gene on the genetic map of the host. If replicates along with the host chromosome and is inherited in the same way as bacterial genes.

In the prophage condition the virus exists in harmony with the host cell and in non infectious. Since the lambda phage normally exists in this condition, it is called a temperate phage. Bacteria containing prophages are called lysogenic bacteria. Viruses whose chromosomes become prophages are called lysogenic viruses. The prophage can be caused to enter the lytic cycle by inducing agents such as ultra violet light) X-rays and mitomycin C. This process is called induction, and results in the release of the prophage from the bacterial chromosome.

DNA and cDNA cloning A number of phage vectors are used in DNA and cDNA cloning. Perhaps the most widely used are phage lambda vectors, which can be used both for cloning of mammalian DNA fragments (genomic DNA libraries) and for the cloning and expression of mammalian cDNAs

(cDNA libraries). The overall cloning strategy is illustrated on the next page. Briefly, genomic DNA is fragmented into pieces of about 15-20 kb in size and then ligated to the "arms" of a pre-digested lambda phage cloning vector. This results in the production of concatenated molecules which are cleaved and packaged into phage heads using commercially available packaging reactions. The packaged phage particles are used to infect susceptible E. coli strains (strains are usually grown in the presence of maltose, since this induces expression of the bacterial lamB gene, which transports maltose into the cell and serves as the receptor for lambda phage). The phage plaques which result from infection of susceptible bacteria can then be screened with radioactive DNA probes or with antibodies, to detect the desired DNA fragment or the protein product of the desired cDNA clone. Important considerations in the use of phage lambda as a cloning vehicle include the following:

1. The vector systems are of the gene-

replacement type. That is, the final recombinant clones remain infectious for E. coli since the

genes which are deleted from the phage are non-essential essential for lytic phage growth. Thus, typical lambda phage vectors are in many ways analogous to certain mammalian virus vectors - such as vaccinia virus or herpesvirus vectors in which a foreign gene has replaced some non-essential viral gene (such as the gene encoding thymidine kinase).

2. The phage's genome cleavage and packaging machinery makes specific nucleolytic

cleavages at the cohesive ends between concatemeric genomes (so called cos sites). This releases the genome unit-length molecules for packaging. Many mammalian viruses also have specific terminal sequences which are important for genome replication and packaging.

3. The wild-type phage genome (i.e., 38-53 kb or so), since the lambda phage head has

a tight constraint on the amount of DNA that it will accomodate. The packaging size limitation of lambda phage vectors is one which is common to most mammalian virus vectors also (i.e., recombinant viral genomes must usually be of wild-type length + 5% or so). Two of the few exceptions are the bacteriophage M13 and mammalian rhabdoviruses (such as vesicular stomatitis virus), both of which can accomodate genomes considerably (>10%) larger than wild-type. This is probably because of the rod-like structure of these viruses (bullet-shape in the case of rhabdoviruses); an increase in the length of the rod allows the particles of these viruses to accomodate an extended genome.

Colony OR Plaque Hybridization for Screening of Libraries

Once a genomic library or cDNA library is available, we may like to use it for isolation of a gene sequence. This can be achieved by colony hybridization technique illustrated. In this

Figure 14: Genomic Library in Lambda Phage

technique, bacteria carrying chimeric vectors are grown into colonies, which are lysed on nitrocellulose filters. Their DNA is denatured in situ and fixed on the filter, which is hybridized with a radioactively labeled probe carrying a sequence related to the gene to be isolated (usually a cloned cDNA for screening of a genomic library).

Colonies carrying this sequence will be identified by dark spots after autoradiography, so that the original chimeric vector carrying the desired gene sequence can be recovered from one or more colonies in the original master plate and used for further experiments. This technique is described as colony hybridization. It is possible that a probe may identify more than one clones or that a gene is fragmented in the library. In such a case, one needs to reconstruct the desired sequence using several overlapping sequences available in the library.

This is a very routine exercise whenever we like to isolate specific DNA sequences from the genome of a species, or from cDNA derived from mRNA of a specific tissue of a species. Sometimes the library may be available not in the form of bacteria transformed with chimeric DNA molecules, but in the form of chimeric phage particles carrying the cloned segments. In such a situation, a bacterial lawn is infected with a mixture of chimeric phage particles (i.e. the library) and a large number of plaques develop overnight. These plaques can be treated just like the colonies in colony hybridization to identify and isolate the chimeric phage particle carrying the gene of interest. This technique is then described as plaque hybridization.

Figure 15: Plaque Hybridization

Cosmid Vectors They have been developed in the late 1970s and have been improved significantly since. (Basic features of a cosmid). They are predominantly plasmids with a bacterial oriV, an antibiotic selection marker and a cloning site, but they carry one, or more recently two cos sites derived from bacteriophage lambda. Depending on the particular aim of the experiment broad host range cosmids, shuttle cosmids or 'mammalian' cosmids (linked to SV40 oriV and mammalian selection markers) are available. The loading capacity of cosmids varies depending on the size of the vector itself but usually lies around 40-45 kb. The cloning procedure involves the generation of two vector arms which are then joined to the foreign DNA. Selection against wildtype cosmid DNA is simply done via size exclusion! Remember however that cosmids always form colonies and not plaques! Also clone density is much lower with around 105 - 106 cfu per ug of ligated DNA. After the construction of recombinant lambda or cosmid libraries the total DNA is transfered into an appropriate E.coli host via a technique called in vitro packaging. The necessary packaging extracts are derived from E.coli cI857 lysogens (red- gam- Sam and Dam (head assembly) and Eam (tail assembly) respectively). These extracts will recognize and package the recombinant molecules in vitro, generating either mature phage particles (lambda-based vectors) or recombinant plasmids contained in phage shells (cosmids). These differences are

reflected in the different infection frequencies seen in favour of lambda-replacement vectors. This compensates for their slightly lower loading capacity. Phage library are also stored and screened easier than cosmid (colonies!) libraries. Target DNA: the genomic DNA to be cloned has to be cut into the appropriate size range of restriction fragments. This is usually done by partial restriction followed by either size fractionation or dephosphorylation (using calf-intestine phosphatase ) in order to avoid chromosome scrambling, ie the ligation of physically unlinked fragments.

Figure 16: Cosmid Vectors

M13 Vectors

Figure 17: M13 vector

M13 Phage as Vectors for DNA Sequencing - M13 is a filamentous bacteriophage of E.

coli and contains a 7.2 kb long single stranded circular DNA. M13 phage has been variously modified to give rise to a M13 mp series of cloning vectors which can be used for cloning of a wide variety of DNA fragments particularly for the purpose of sequencing through Sanger's method of dideoxy chain termination. Cloning vectors of M13 mp series have a lac Z gene that complements gal host (e.g. JM 103 or JM 104) giving blue colonies. But if a foreign DNA segment is inserted at one of the polylinker sites associated with lac Z gene, it inactivates lac Z gene and no complementation is possible.

Thus on transformation, only white or clear plaques are obtained thus permitting selection of recombinant M13mp plaques. Once the foreign DNA is cloned in M13 vector, commercially available oligonucleotide primers are used for copying the insert in presence of dideoxynucleotides, so that fragments of different sizes with known termini are produced, permitting the determination of the sequence. Reversed order of the restriction sites in polylinker is present in a pair of vectors like M13mp8 and M13mp9 permitting sequencing from both the ends of double stranded DNA molecule. The extension of the commercial primer or cloned fragment in Mil vector in the presence of radioactively labelled nucleotides will also allow generation of radioactive single stranded probes

Complementary DNA (cDNA) In genetics, complementary DNA (cDNA) is DNA synthesized from a mature mRNA

template in a reaction catalyzed by the enzyme reverse transcriptase and the enzyme DNA

polymerase. cDNA is often used to clone eukaryotic genes in prokaryotes. When scientists

want to express a specific protein in a cell that does not normally express that protein (i.e.,

heterologous expression), they will transfer the cDNA that codes for the protein to the

recipient cell. cDNA is also produced by retroviruses (such as HIV-1, HIV-2, Simian

Immunodeficiency Virus, etc.) which is integrated into its host to create a provirus.

Figure 18: Purification of cDNA on a Oligo dT column

Complementary DNA (cDNA) In genetics, complementary DNA (cDNA) is DNA synthesized from a mature mRNA template

in a reaction catalyzed by the enzyme reverse transcriptase and the enzyme DNA

polymerase. cDNA is often used to clone eukaryotic genes in prokaryotes. When scientists

want to express a specific protein in a cell that does not normally express that protein (i.e.,

heterologous expression), they will transfer the cDNA that codes for the protein to the

recipient cell. cDNA is also produced by retroviruses (such as HIV-1, HIV-2, Simian

Immunodeficiency Virus, etc.) which is integrated into its host to create a provirus.

Figure 19: cDNA synthesis

Phagemid

A phagemid or phasmid is a type of cloning vector developed as a hybrid of the filamentous

phage M13 and plasmids to produce a vector that can grow as a plasmid, and also be

packaged as single stranded DNA in viral particles. Phagemids contain an origin of

replication (ori) for double stranded

replication, as well as an f1 ori to

enable single stranded replication and

packaging into phage particles. Many

commonly used plasmids contain an f1

ori and are thus phagemids. Similarly to

a plasmid, a phagemid can be used to

clone DNA fragments and be

introduced into a bacterial host by a

range of techniques (transformation,

electroporation). However, infection of a

bacterial host containing a phagemid

with a 'helper' phage, for example

VCSM13 or M13K07, provides the

necessary viral components to

enable single stranded DNA

replication and packaging of the phagemid DNA into phage particles. These are secreted

through the cell wall and released into the medium. Filamentous phage retard bacterial

growth but, in contrast to lambda and T7 phage, are not generally lytic. Helper phage are

usually engineered to package less efficiently than the phagemid so that the resultant phage

particles contain predominantly phagemid DNA. F1 Filamentous phage infection requires the

presence of a pilus so only bacterial hosts containing the F-plasmid or its derivatives can be

used to generate phage particles. Prior to the development of cycle sequencing phagemids

were used to generate single stranded DNA template for sequencing purposes. Today

phagemids are still useful for generating templates for site-directed mutagenesis. Detailed

characterisation of the filamentous phage life cycle and structural features lead to the

development of phage display technology, in which a range of peptides and proteins can be

expressed as fusions to phage coat proteins and displayed on the viral surface. The

displayed peptides and polypeptides are associated with the corresponding coding DNA

within the phage particle and so this technique lends itself to the study of protein-protein

interactions and other ligand/receptor combinations.

Figure 20: Phagemid

References: Lambda Phage vector animation:

http://www.blackwellpublishing.com/wagner/animations/lambdaw.html

1. S. Shuman, Journal of Biological Chemistry 284, 17365 (2009).