Chapter 4 Molecular Cloning Methods

4.1 Gene Cloning Gene cloning is an indispensable molecular biology technique that allows scientists to produce large quantities of their gene of interest Gene cloning links eukaryotic genes to small bacterial or phage DNAs and inserting these recombinant molecules into bacterial hosts Gene cloning can produce large quantities of these genes in pure form

The Role of Restriction Endonucleases Restriction endonucleases, first discovered in the late 1960s in E. coli, are named for preventing invasion by foreign DNA by cutting it into pieces These enzymes cut at sites within the foreign DNA instead of chewing from the ends By cutting DNA at specific sites they function as finely honed molecular knives

Naming Restriction Endonucleases Restriction endonucleases are named using the 1st three letters of their name from the Latin name of their source microorganism Hind III First letter is from the genus H from Haemophilus Next two letters are the 1st two letters of the species name in from influenzae Sometimes the strain designation is included “d” from strain Rd If microorganism produces only 1 restriction enzyme, end the name with Roman numeral I Hind I If more than one restriction enzyme is produced, the others are numbered sequentially II, III, IV, etc.

Restriction Endonuclease Specificity Restriction endonucleases recognize a specific DNA sequence, cutting ONLY at that sequence They recognize 4-bp, 6-bp, 8-bp palindromic sequences The frequency of cuts lessens as the recognition sequence is longer They cut DNA reproducibly in the same place

Restriction-Modification System What prevents these enzymes from cutting up the host DNA? They are paired with methylases Theses enzymes recognize, methylate the same site Together they are called a restriction-modification system, R-M system Methylation protects DNA, after replication the parental strand is already methylated

An Experiment Using Restriction Endonuclease: Boyer and Cohen An early experiment used EcoRI to cut 2 plasmids, small circular pieces of DNA independent of the host chromosome Each plasmid had 1 EcoRI site Cutting converted circular plasmids into linear DNA with the same sticky ends The ends base pair Some ends re-close Others join the 2 pieces DNA ligase joins 2 pieces with covalent bonds

Vectors Vectors function as DNA carriers to allow replication of recombinant DNAs Typical experiment uses 1 vector plus a piece of foreign DNA The inserted and foreign DNA depends on the vector for its replication as it does not have an origin of replication, the site where DNA replication begins There are 2 major classes of vectors: Plasmids Phages

Plasmids As Vectors pBR plasmids were developed early but are rarely used today pUC series is similar to pBR 40% of the DNA has been deleted Cloning sites are clustered together into one area called the multiple cloning site (MCS) MCS allows one to cut the vector and foreign gene with two different restriction enzymes and use a directional cloning technique to know the orientation of the insert

Screening: antibiotics and b-galactosidase Screening capabilities within plasmids: Antibiotic resistance genes (i.e., ampicillin resistance gene) allow for the selection of bacteria that have received a copy of the vector Multiple cloning site inserted into the gene lacZ’ coding for the enzyme b-galactosidase Clones with foreign DNA in the MCS disrupt the ability of the cells to make b-galactosidase Plate on media with a b-galactosidase indicator (X-gal) and clones with intact b-galactosidase enzyme will produce blue colonies Colorless colonies should contain the plasmid with foreign DNA compared to blue colonies that do not contain the plasmid with DNA

Phages As Vectors Bacteriophages are natural vectors that transduce bacterial DNA from one cell to another Phage vectors infect cells much more efficiently than plasmids transform cells Clones are not colonies of cells using phage vectors, but rather plaques, a clearing of the bacterial lawn due to phage killing the bacteria in that area

l Phage Vectors First phage vectors were constructed by Fred Blattner and colleagues Modifications included removal of the middle region and retention of the genes needed for phage replication Could replace removed phage genes with foreign DNA Advantage: Phage vectors can receive larger amounts of foreign DNA (up to 20kb of DNA) Traditional plasmid vectors take much less Phage vectors require a minimum size foreign DNA piece (12 kb) inserted to package into a phage particle

Cloning Using a Phage Vector

Genomic Libraries A genomic library contains clones of all the genes from a species genome Restriction fragments of a genome can be packaged into phage using about 16 – 20 kb per fragment This fragment size will include the entirety of most eukaryotic genes Once a library is established, it can be used to search for any gene of interest

Selection via Plaque Hybridization Searching a genomic library requires a probe to determine which clone contains the desired gene Ideal probe – labeled nucleic acid with sequence matching the gene of interest

Cosmids Cosmids are designed for cloning large DNA fragments Behave both as plasmid and phage and contain cos sites, cohesive ends of phage DNA that allow the DNA to be packaged into a l phage head Plasmid origin of replication permitting replication as plasmid in bacteria Nearly all l genome removed so there is room for large inserts (40-50 kb) Very little phage DNA yields them unable to replicate, but they are infectious and carry their recombinant DNA into bacterial cells

M13 Phage Vectors Long, thin, filamentous phage Contains: Advantage Gene fragment with b-galactosidase Multiple cloning site like the pUC family Advantage This phage’s genome is single-stranded DNA Fragments cloned into it will be recovered in single-stranded form

M13 Cloning to Recover Single-stranded DNA Product After infecting E. coli cells, single-stranded phage DNA is converted to double-stranded replicative form (RF) Use the replicative form for cloning foreign DNA into MCS Recombinant DNA infects host cells resulting in single-stranded recombinant DNA Phage particles, containing single-stranded phage DNA is secreted from transformed cells and can be collected from media

Phagemids Phagemids are also vectors Like cosmids have aspects of both phages and plasmids Has MCS inserted into lacZ’ gene to screen blue/ white colonies Has origin of replication of single-stranded phage f1 to permit recovery of single-stranded recombinant DNA MCS has 2 phage RNA polymerase promoters, 1 on each side of MCS

Eukaryotic Vectors and Very High Capacity Vectors There are vectors designed for cloning genes into eukaryotic cells Other vectors are based on the Ti plasmid to carry genes into plant cells Yeast artificial chromosomes (YAC) and bacterial artificial chromosomes (BAC) are used for cloning huge pieces of DNA

Identifying a Specific Clone With a Specific Probe Probes are used to identify a desired clone from among the thousands of irrelevant ones Two types are widely used Polynucleotides (also called oligonucleotides) Antibodies

Polynucleotide Probes Looking for the gene you want, you might use the homologous gene from another organism If already cloned and there is enough sequence similarity to permit hybridization Need to lower stringency of hybridization conditions to tolerate some mismatches High temperature, high organic solvent concentration and low salt concentration are factors that promote separation of two strands in a DNA double helix and can be adjusted as needed

Protein-based Polynucleotide Probes No homologous DNA from another organism? If amino acid sequence is known, deduce a set of nucleotide sequences to code for these amino acids Construct these nucleotide sequences chemically using the synthetic probes Why use several? Genetic code is degenerate with most amino acids having more than 1 nucleic acid triplet Must construct several different nucleotide sequences for most amino acids

cDNA Cloning cDNA - complementary DNA or copy DNA that is a DNA copy of RNA A cDNA library is a set of clones representing as many as possible of the mRNAs in a given cell type at a given time Such a library can contain tens of thousands of different clones

Making a cDNA Library

Reverse Transcriptase Central to successful cloning is the synthesis of cDNA from an mRNA template using reverse transcriptase (RT), an RNA-dependent DNA polymerase RT cannot initiate DNA synthesis without a primer Use the poly(A) tail at 3’ end of most eukaryotic mRNA so that oligo(dT) may serve as primer

Ribonuclease H RT with oligo(dT) primer has made a single-stranded DNA off of mRNA Need to remove the RNA Partially degrade the mRNA using ribonuclease H (RNase H) Enzyme degrades RNA strand of an RNA-DNA hybrid Remaining RNA fragments serve as primers for “second strand” DNA using nick translation

Nick Translation The nick translation process simultaneously: Removes DNA ahead of a nick Synthesizes DNA behind nick Net result moves the nick in the 5’ to 3’ direction Enzyme often used is E. coli DNA polymerase I Has 5’ to 3’ exonuclease activity Allows enzyme to degrade DNA ahead of the nick

Terminal Transferase cDNAs don’t have the sticky ends of genomic DNA cleaved with restriction enzymes Blunt ends will ligate, but is inefficient Generate sticky ends using enzyme terminal deoxynucleotidyl transferase (TdT), terminal transferase with one dNTP If use dCTP with the enzyme dCMPs are added one at a time to 3’ ends of the cDNA Same technique adds oligo(dG) ends to vector Generate ligation product ready for transformation

Rapid Amplification of cDNA Ends If generated cDNA is not full-length, missing pieces can be filled in using rapid amplification of cDNA ends (RACE) Technique can be used to fill in either the missing portion at the 5’-end (usual problem) Analogous technique can be used to fill in a missing 3’-end

RACE Procedure Use RNA prep containing mRNA of interest and the partial cDNA Anneal mRNA with the incomplete cDNA Reverse transcriptase will copy rest of the mRNA Tail the completed cDNA with terminal transferase using oligo(dC) Second strand synthesis primed with oligo(dG)

4.2 The Polymerase Chain Reaction Polymerase chain reaction (PCR) is used to amplify DNA and can be used to yield a DNA fragment for cloning Invented by Kary Mullis and colleagues in 1980s Special heat-stable polymerases are now used that are able to work after high temperatures - researchers no longer need to add fresh DNA polymerase after each round of replication

Standard PCR Use enzyme DNA polymerase to copy a selected region of DNA Add short pieces of DNA (primers) that hybridize to DNA sequences on either side of piece of interest – causes initiation of DNA synthesis through that area, X Copies of both strands of X and original DNA strands are templates for the next round of DNA synthesis The selected region of DNA now doubles in amount with each synthesis cycle

Amplifying DNA by PCR

Using Reverse Transcriptase PCR (RT-PCR) in cDNA Cloning To clone a cDNA from just one mRNA whose sequence is known, a type of PCR called reverse transcriptase PCR (RT-PCR) can be used Difference between PCR and RT-PCR Starts with an mRNA, not dsDNA Begin by converting mRNA to DNA Use forward primers to convert ssDNA to dsDNA Continue with standard PCR

RT-PCR to clone a single cDNA With care, restriction enzyme sites can even be added to the ends of the cDNA of interest Able to generate sticky ends for ligation into vector of choice 2 sticky ends permits directional cloning

Real-Time PCR Real-time PCR quantifies the amplification of the DNA as it occurs As the DNA strands separate, they anneal to forward and reverse primers, and to a fluorescent-tagged oligonucleotide complementary to part of one DNA strand that serves as a reporter probe

Real-Time PCR A fluorescent-tagged oligonucleotide serves as a reporter probe Fluorescent tag at 5’-end Fluorescence quenching tag at 3’-end As PCR progresses from the forward primer the 5’ tag is separated from the 3’ tag and allows the 5’ tag to fluoresce Fluorescence increases with incorporation into DNA product and can be quantitated

4.3 Methods of Expressing Cloned Genes Cloning a gene permits Production of large quantities of a particular DNA sequence for detailed study Large quantities of the gene’s product can also be obtained for further use Study Commerce

Expression Vectors Vectors discussed so far are used to first put a foreign DNA into a bacterium to replicate and screen Expression vectors are those that can yield protein products of the cloned genes Bacterial expression vectors typically have two elements required for active gene expression; a strong promoter and a ribosome binding site near an initiating codon

Fusion Proteins Some cloning vectors, pUC and pBS, can work as expression vectors using lac promoter If inserted DNA is in the same reading frame as interrupted gene, a fusion protein results These have a partial b-galactosidase sequence at amino end Inserted cDNA protein sequence at carboxyl end

Inducible Expression Vectors Main function of expression vector is to yield the product of a gene – usually more is better For this reason, expression vectors have very strong promoters It is usually advantageous to keep a cloned gene repressed until time to express Large quantities of eukaryotic protein in bacteria are usually toxic Can accumulate to levels that interfere with bacterial growth Expressed protein may form insoluble aggregates, called inclusion bodies

Controlling the lac Promoter lac promoter is somewhat inducible Stays off until stimulated by inducer IPTG However, repression is typically incomplete or leaky and some expression will still occur To avoid this problem, use a plasmid or phagemid carrying its own lacI repressor gene to keep the cloned gene off until it is induced by IPTG

Alternatives to the lac Promoter The hybrid trc promoter combines the strength of the trp (tryptophan operon) promoter with the inducibility of the lac promoter Promoter from ara operon, PBAD, allow fine control of transcription Inducible by arabinose, a sugar Transcription rate varies with arabinose concentration

Alternatives to the lac Promoter The lambda (l) phage promoter, PL, is tightly controlled Expression vectors with this promoter-operator system are used in host cells with temperature-sensitive l repressor gene Repressor functions at low temperatures Raise temperature above the nonpermissive level (42’C) and the repressor doesn’t function and the cloned gene is expressed

Expression Vectors That Produce Fusion Proteins Most vectors express fusion proteins The actual natural product of the gene isn’t made Extra amino acids help in purifying the protein product Oligohistidine expression vector has a short sequence just upstream of MCS encoding 6 His Oligohistidine has a high affinity for divalent metal ions like nickel (Ni2+) Permits purification by nickel affinity chromatography The his tag can be removed using enzyme enterokinase without damage to the protein product

Using an Oligohistidine Expression Vector

Expression vector lgt11 This phage contains the lac control region followed by the lacZ gene The cloning sites are located within the lacZ gene Products of gene correctly inserted will be fusion proteins with a b-galactosidase leader

Detecting positive lgt11 clones via antibody screening Lambda phages with cDNA inserts are plated Protein released are blotted onto a support Probe with antibody specific to protein Antibody bound to protein from plaque is detected with labeled protein A

Bacterial Expression System Shortcomings There are problems with expression of eukaryotic proteins in a bacterial system Bacteria may recognize the proteins as foreign and destroy them Post-translational modifications are different in bacteria Bacterial environment may not permit correct protein folding Very high levels of cloned eukaryotic proteins can be expressed in useless, insoluble form

Eukaryotic Expression Systems Avoid bacterial expression problems by expressing the protein in a eukaryotic cell Initial cloning done in E. coli using a shuttle vector, able to replicate in both bacterial and eukaryotic cells Yeast is suited for this purpose Rapid growth and ease of culture A eukaryote with more appropriate post-translational modification Use of the yeast export signal peptide secretes protein into growth medium for easy purification

Use of Baculovirus As Expression Vector Viruses in this class have a large circular DNA genome, 130 kb Major viral structural protein is made in huge amounts in infected cells The promoter for this protein, polyhedrin, is very active These vectors can produce up to 0.5 g of protein per liter of medium Nonrecombinant viral DNA entering cells does not result in infectious virus as it lacks an essential gene supplied by the vector

Expressing a Gene in a Baculovirus

Animal Cell Transfection Carried out in two ways: Calcium phosphate Mix cells with DNA in a phosphate buffer and add a solution of calcium salt to form a precipitate The cells take up the calcium phosphate crystals, which include some DNA Liposomes The DNA is mixed with lipid to form liposomes, small vesicles with some of the DNA inside DNA-bearing liposomes fuse with the cell membrane to deliver DNA inside the cell

Using the Ti Plasmid to Transfer Genes to Plants Genes can be introduced into plants with vectors that can replicate in plant cells Common bacterial vector promoters and replication origins are not recognized by plant cells Plasmids are used containing T-DNA T-DNA is derived from a plasmid known as tumor-inducing (Ti) Ti plasmid comes from bacteria that cause plant tumors called crown galls

Ti Plasmid Infection Bacterium infects plant, transfers Ti plasmid to host cells T-DNA integrates into the plant DNA causing abnormal proliferation of plant cells T-DNA genes direct the synthesis of unusual organic acids, opines which can serve as an energy source to the infecting bacteria but are useless to the plant

The Ti Plasmid Transfers Crown Gall

Use of the T-DNA Plasmid