• Recombinant DNA technology depends on the ability to producelarge numbers of identical DNA molecules (clones).

• Clones are typically generated by placing a DNA fragment ofinterest into a vector DNA molecule, which can replicate in ahost cell.

• When a single vector containing a single DNA fragment isintroduced into a host cell, large numbers of this fragment arereproduced along with the vector.

• Two commonly used vectors for cloning are E. coli plasmidvectors and bacteriophage λ vectors

DNA restriction cutting, plasmid, phage, DNA ligation,antibiotic selection, cDNA, cDNA Library, expression vector

Molecular Cloning

Lecture 13

Ligase reaction: opposite to restriction enzyme, requires 5’-P (from DNA2) and 3’-OH (from DNA1).

Ligase mechanism:

DNA ligase forms activatedligase-AMP (from ATP or NAD)�,AMP links to 5’-P, 3’-OHattacks forming newphosphodiester bond, seal!

Ligation of the sticky ends:two DNA molecules, cleaved withEcoRI and ligate to formrecombinant molecules

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

Formation of cohesive ends

Plasmid: circular, ds, extrachromosomal self-replicating DNA molecule occuring naturally inbacteria, yeast and higher eukaryotic cells, either parasitic or symbiotic, ~kb to 100 kb, at leastone to the daughter cell (drug-resistance, transfer genes encoding proteins formingmacromolecular tube or pilus).

Plasmid and cloning vector

Cloning vector: engineered plasmids with reduced size (~3 kb), containing only ori, drug-resistance gene, cloning site. Ampr encodes β-lactamase which inactivates ampicillin.

cut both of the vector and insert with BamH1,

vector treated with alkaline phosphatase(prevent resealing),

ligase put insert into the vector, two nicks will besealed in vivo.

Basic cloning

Recombinant DNA

The plasmid and theforeign DNA are cut by arestriction endonuclease(EcoRI in this example)producing intermediateswith sticky andcomplementary ends.Those two intermediatesrecombine by base-pairing and are linked bythe action of DNA ligase.A new plasmid containingthe foreign DNA as aninsert is obtained. A fewmismatches occur,producing an undesirablerecombinant.

First recombinant DNA produced:Stanley Cohen and Herbert Boyer cut 2 plasmids withEcoRI, ligate and screen for E.coli clones that areresistant to both antibiotics since they harbor therecombinant plasmids. Patent 4,237,224, 1980

http://www.dnai.org/text/mediashowcase/index2.html?id=1186

1973 PNAS 70, 3240

pBR322 plasmidHost containing pBR322 with insert at different restriction sites can be selected.

There are many sites can be used.

Make construct, transformbacteria, screen fortetracycline-resistant butampicillin-sensitive clonesusing replica plating (make areplica plate treating withampicillin, sensitive clonesare recovered from theoriginal plate).

Cloning at the PstI site

Chemically synthesized polylinkercontaining one copy of severaldifferent restriction sites isintroduced into the vector tofacilitate directional cloning.

Polylinkers

MCS is inserted into a gene encoding the N-terminal part of β-galactosidase.Clones harboring the vector plus an insert remain white.

pUC series: derived from pBR322 (40% deleted), the MCS is in the lacZ’ (N-terminal of β-galactosidase, the host contains the C-terminus portion, MCS startsafter the 7th codon, retains the ORF), so no insert blue (with X-gal), with insertwhite.

MCS: multiple cloning site

The general procedure for cloning with plasmid vectors:plasmid with Ampr and insert is transformed into host withCaCl2 or other methods, grow in ampicillin plate, survivingcells form colony.

Plasmid cloning permits isolation of DNA fragments fromcomplex mixtures: Each colony is derived from a singlecell containing the same plasmid.

Molecular cloning

Process by which a plasmid is used to import recombinant DNA into a host cell for cloning.

In DNA cloning, a DNA fragment that contains a gene of interest is inserted into a cloningvector or plasmid. The plasmid carrying genes for antibiotic resistance, and a DNA strand,which contains the gene of interest, are both cut with the same restriction endonuclease.The plasmid is opened up and the gene is freed from its parent DNA strand. They havecomplementary "sticky ends." The opened plasmid and the freed gene are mixed withDNA ligase, which reforms the two pieces as recombinant DNA.

This recombinant DNA stew is allowed to transform a bacterial culture, which is thenexposed to antibiotics. All the cells except those which have been encoded by the plasmidDNA recombinant are killed, leaving a cell culture containing the desired recombinantDNA.

DNA cloning allows a copy of any specific part of a DNA (or RNA) sequence to beselected among many others and produced in an unlimited amount. This technique is thefirst stage of most of the genetic engineering experiments: production of DNA libraries,PCR, DNA sequencing, et al.

Cloning strategy

Molecular cloning

Molecular cloning

DNA Libraries in λ phage and othercloning vectors

• Cloning all of the genomic DNA of higher organismsinto plasmid vectors is not practical due to therelatively low transformation efficiency of E. coli andthe small number of transformed colonies that can begrown on a typical culture plate

• Cloning vectors derived from bacteriophage do notsuffer from such limitations

• A collection of clones that includes all the DNAsequences of a given species is called a genomiclibrary

• A genomic library can be screened for clonescontaining a sequence of interest

(i) EM of bacteriophage λ virion.(ii) Simplified view of bacteriophage genome:about 60 genes, replaceable regionnot essential and can be replaced by foreign DNA up to 25 kb. Large segment ofthe 48kb DNA of the λ phage are not essential for productive infection and can bereplaced with inserts.(iii)Assembly of bacteriophage λ virion: during late stage of λ infectionconcatomers are formed, Nu 1 and A protein push 1 copy of λ from concatomerinto the head, then tail added and they are ready to go to the war.

cloning

Charon phage 4 takes 20 kb,

remove the stuffer,

ligate the insert,

mix with the in vitropackaging extract,

infect cells,

the insert has to be between12 kb and 20 kb.

λ phage vector

Alternative infection modes for λ phage: lytic or lysogenic pathway

A set of lambda (or plasmid)clones that collectivelycontain every DNAsequence in the genome ofa particular organism.

Nearly complete genomiclibraries of higherorganisms can be preparedby lambda cloning.

Genomic Library

(i) A set of lambda (or plasmid) clones thatcollectively contain every DNA sequence inthe genome of a particular organism. Nearlycomplete genomic libraries of higherorganisms can be prepared by lambdacloning.

(ii) Construction of a genomic library:Sau3A and BamH1 generatecomplementary sticky ends.

(iii) Plaque hybridization: selection of positiveclones, filter replicates the dish, plaque containingphage DNA denatured by base, block the filter with non-specific DNA or protein, hybridize with specific geneprobe, autoradiography to select the target .gene.

(iv) The most common approach to identifying aspecific clone involves screening a library byhybridization with radioactively labeled DNA orRNA probes. Identification of a specific clonefrom a l phage library by membranehybridization. Identifying, analyzing, andsequencing cloned DNA

Larger DNA fragments up to 45kb can be cloned in cosmids andother vectors,

cosmid has cohesive ends forpackaging and plasmid ori forreplication as plasmids inbacteria.

cosmids cannot replicate asphages but they are stillinfectious.

Cosmid vector

M13 Phage: a filamentousvirus 900 nm long and 9 nmwide, single strand 6.4kbcircle DNA protected by 2710identical proteins, gets into E.coli via sex pilus, replicate toRF, only (+) is packed intovirus particle. Useful forsequencing.

M13 Phage

oligo-dT column to purify mRNA

Complementary DNA (cDNA) librariesare prepared from isolatedmRNAs

Complementary DNA

Formation of cDNA duplex: RT, alkali digestion, oligo(dG) tailing, oligo(dC) primer

Complementary DNA (cDNA) libraries

Preparation of a cDNA library

Making a cDNA Library: ReverseTranscriptase, RNase H (degrade RNA inRNA-DNA hybrid) , DNA polymerase I (usingRNA as primer and perform nick translation),Terminal deoxynucleotidyl transferase (addingdCTP to the cDNA duplex, and dGTP to thevector), Ligase and pol I in the host performligation, RNA removal, and sealing.

Making a cDNA Library

Use RT-PCR to clone asingle cDNA if the sequenceof mRNA is known.

pBS or pBluescript, MCS inserted into lacZ’, ori of the ss phage f1, T3 and T7 phageRNA polymerase promoters.

Phagemid

(i) Forming fusion protein in plasmid vector: pUC and pBS vectors place insertedDNA under the control of the lac promoter. If the DNA is in frame with the lacZ’gene, a fusion protein will be expressed.

The expression vector contains a fragment of the E. coli chromosome containing thelac promoter and the neighboring lacZ gene. In the presence of the lactose analogIPTG, RNA polnormally transcribes the lacZ gene, producing lacZ mRNA, which istranslated to the encoded protein, β-galactosidase.

Expression vector

The lacZ gene can be cut out of the expression vector with restriction enzymesand replaced by the G-CSF cDNA. After transformation, IPTG will induce theexpression of G-CSF protein.

E. coli expression systems canproduce full-length proteins:Producing high levels of proteinsfrom cloned cDNAs. Manyproteins are normally expressed atvery low concentrations withincells, which makes isolation ofsufficient amounts for analysisdifficult. To overcome thisproblem, DNA expression vectorscan be used to produce largeamounts of full length proteins.Lac promoter.

Even larger amounts of a desired protein can be expressed with a two-stepsystem, 10-70% of the total protein synthesized by these cells after IPTG is theprotein of interest.

Two-step system : T7 RNA polymerase and T7 late promoter

Forming fusion protein in phage vector: λg11with lac control region followed by lacZ gene,cloning sites are located within the lacZ gene, direct detection of protein with antibody.

Degenerate probe: screen for the correct cDNA or genomicclone from the library.