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Molecular Cloning Methods Chapter 4 Vectors • Vectors - DNA carriers to allow replication of recombinant DNAs • Typical experiment uses 1 vector plus a piece of foreign DNA – Depends on the vector for its replication – Foreign DNA has no origin of replication • There are 2 major classes of vectors: – Plasmids – Phages pBR322 Plasmid • pBR322 - cloning methods • Resistance for 2 antibiotics • Tetracycline • Ampicillin – Origin of replication between the 2 resistance genes – Only 1 site for several restriction enzymes Plasmids As Vectors • pUC series is similar to pBR – 40% of the DNA - including tetracycline resistance has been deleted – Cloning sites are clustered together into one area called the multiple cloning site (MCS) pBR322 Cloning Clone a foreign DNA into the PstI site of pBR322 • Cut the vector to generate the sticky ends • Cut foreign DNA with PstI also – compatible ends • Combine vector and foreign DNA with DNA ligase to seal sticky ends • Now transform the plasmid into E. coli Bacterial Transformation • Traditional method - incubating bacterial cells in concentrated calcium salt solution – The solution makes the cell membrane permeable to the plasmid DNA • Newer method - high voltage to drive the DNA into the cells - electroporation Screening Transformants • Transformation produces bacteria with: – Religated plasmid – Religated insert – Recombinants • Identify the recombinants using the antibiotic resistance – Grow cells with tetracycline so only cells with plasmid grow - not foreign DNA only Screening With Replica Plating • Replica plating transfers clone copies from original tetracycline plate to a plate containing ampicillin • Desired colonies are those that do NOT grow on the new ampicillin plate pUC and β-galactosidase Newer pUC plasmids have: – Ampicillin resistance gene – Multiple cloning site inserted into the gene lacZ’ coding for the enzyme β-galactosidase • Clones with foreign DNA in the MCS disrupt the ability of the cells to make β-galactosidase • Plate on media with a β-galactosidase indicator (X-gal) and clones with intact β-galactosidase enzyme will produce blue colonies • Colorless colonies should contain the plasmid with foreign DNA Directional Cloning • Cut a plasmid with 2 restriction enzymes from the MCS • Clone in a piece of foreign DNA with 1 sticky end recognizing each enzyme • The insert DNA is placed into the vector in only 1 orientation • Vector religation is also prevented as the two restriction sites are incompatible 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 – plaques - a clearing of the bacterial lawn due to phage killing the bacteria in that area Phage Vectors • First phage vectors were constructed by Fred Blattner and colleagues – Removed middle region – Replaced with foreign DNA – Retained genes needed for phage replication • Charon phage • General term - replacement vectors Cloning Using a Phage Vector • Phage vectors can receive larger amounts of foreign 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 Genomic Libraries • A genomic library contains clones of all the genes from a genome • Restriction fragments of a genome can be packaged into phage using about 16 – 20 kb per fragment • Once a library is established - it can be used to search for any gene of interest Plaque Hybridization • Searching a genomic library requires probe showing which clone contains desired gene • Ideal probe – labeled nucleic acid with sequence matching the gene of interest Homework • Explain Cosmids, M13 phage vectors and Phagemids and Eukaryotic vectors and very high capacity vectors– their features and functions 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 • Use homologous gene from another organism – If already cloned – Enough sequence similarity to permit hybridization – Need to lower stringency of hybridization conditions to tolerate some mismatches Control of Hybridization Stringency • Adjust conditions until only perfectly matched DNA strands form a duplex = high stringency • Lowering these conditions lowers stringency until DNA strands with a few mismatches can hybridize Protein-based Polynucleotide Probes • 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 • A cDNA library is a set of clones representing many mRNAs in a given cell type at a given time – Such a library can contain tens of thousands of different clones – One cDNA – a clone containing a DNA copy of just one mRNA Making a cDNA library Role of RT in making a cDNA library • Synthesis of cDNA from an mRNA template using reverse transcriptase (RT) – 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 Role of Rnase H in making a cDNA library • Partially degrade the mRNA using ribonuclease H (RNase H) – Enzyme degrades RNA strand of an RNADNA hybrid – Remaining RNA fragments serve as primers for “second strand” DNA using nick translation Nick translation – DNA polymerase I shows 5’ to 3’ exonuclease activity – Removes/degrades DNA ahead of a nick – Synthesizes DNA behind nick – Net result moves or translates the nick in the 5’ to 3’ direction Role of terminal transferase in making a cDNA library • Generate sticky ends using terminal deoxynucleotidyl transferase (TdT), terminal transferase with one dNTP – 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 How to detect positive clones? • Plasmid or phagemid like pUC or pBS will be used with colony hybridization and a labeled DNA probe 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) or fill in a missing 3’-end 5’-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) Polymerase Chain Reaction • Polymerase chain reaction (PCR) can yield a DNA fragment for cloning • Invented by Kary Mullis and colleagues in 1980s • PCR is: – More recently developed – Very useful for cloning cDNAs PCR Reverse Transcriptase PCR (RT-PCR) in cDNA cloning • To clone a cDNA from just one mRNA whose sequence is known - reverse transcriptase PCR (RT-PCR) Reverse Transcriptase PCR (RT-PCR) in cDNA cloning • Difference between PCR and RT-PCR – Start with an mRNA not double-stranded DNA – Begin by converting mRNA to DNA – Next use forward primer to convert ssDNA to dsDNA – Now standard PCR continues RT-PCR Generates Sticky Ends • Restriction enzyme sites can be added to the cDNA of interest • Able to generate sticky ends for ligation into vector of choice • 2 sticky ends permits directional cloning Fluorescent Tags in Real-Time PCR • Fluorescent-tagged oligonucleotide serves as a reporter probe – Fluorescent tag at 5’end – Fluorescence quenching tag at 3’end • With PCR rounds the 5’ tag is separated from the 3’ tag • Fluorescence increases with incorporation into DNA product Methods of Expressing Cloned Genes • Expression vectors are those that can yield protein products of the cloned genes – For high level expression of a cloned gene – Bacterial vectors have a strong promoter and a ribosome binding site near ATG codon Inducible expression vectors • Prefer 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, inclusion bodies Expression vectors produce fusion proteins Expression vectors 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 Ni2+ – Permits purification by nickel affinity chromatography – His tag can be removed using enzyme enterokinase without damage to the protein product Bacterial Expression System Shortcomings – Problems - Bacteria may recognize the proteins as foreign and destroy them – Posttranslational 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 How to avoid bacterial expression problems? • Initial cloning done in E. coli using a shuttle vector - replicate in both bacterial and eukaryotic cells • Yeast is suited for this purpose – Rapid growth and ease of culture – Eukaryote with more appropriate posttranslational modification – Secretes protein in growth medium - 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 – Promoter for polyhedrin protein is very active – These vectors can produce up to 0.5 g of protein per liter of medium Use of Baculovirus As Expression Vector Formation of crown gall tumors Agrobacterium tumefaciens • Bacterium infects plant - transfers Ti plasmid to host cells • T-DNA integrates into the plant DNA causing abnormal proliferation of plant cells – galls/tumors • 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 Using Ti-plasmid to transfer genes to plants • • • This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60). 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