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Download Part II: Recombinant DNA Technology
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Recombinant DNA Technology Definition of recombinant DNA Production of a unique DNA molecule by joining together two or more DNA fragments not normally associated with each other DNA fragments are usually derived from different biological sources Applications Gene isolation/purification/synthesis Sequencing/Genomics/Proteomics Polymerase chain reaction (PCR) Mutagenesis (reverse genetics) Expression analyses (transcriptional and translational levels) Restriction fragment length polymorphisms (RFLPs) Biochemistry/ Molecular modeling High throughput screening Combinatorial chemistry Gene therapy Recombinant Vaccines Genetically modified crops Biosensors Monoclonal antibodies Cell/tissue culture Xenotransplantation Bioremediation Production of next generation antibiotics Forensics Bioterrorism detection Common steps involved in isolating a particular DNA fragment from a complex mixture of DNA fragments or molecules 1. DNA molecules are digested with enzymes called restriction endonucleases which reduces the size of the fragments Renders them more manageable for cloning purposes 2. These products of digestion are inserted into a DNA molecule called a vector Enables desired fragment to be replicated in cell culture to very high levels in a given cell 3. Introduction of recombinant DNA molecule into an appropriate host cell Transformation or transfection Each cell receiving rDNA = CLONE May have thousands of copies of rDNA molecules/cell after DNA replication As host cell divides, rDNA partitioned into daughter cells 4. Population of cells of a given clone is expanded, and therefore so is the rDNA. Amplification DNA can be extracted, purified and used for molecular analyses Investigate organization of genes Structure/function Activation Processing Gene product encoded by that rDNA can be characterized or modified through mutational experiments II. Restriction Endonucleases A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded or single stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses. Inside a bacterial host, the restriction enzymes selectively cut up foreign DNA in a process called restriction; host DNA is methylated by a modification enzyme (a methylase) to protect it from the restriction enzyme’s activity. Collectively, these two processes form the restriction modification system. To cut the DNA, a restriction enzyme makes two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix. Restriction enzymes recognize a specific sequence of nucleotides and produce a double-stranded cut in the DNA. While recognition sequences vary between 4 and 8 nucleotides, many of them are palindromic, which correspond to nitrogenous base sequences that read the same backwards and forwards. In theory, there are two types of palindromic sequences that can be possible in DNA. The mirror-like palindrome is similar to those found in ordinary text, in which a sequence reads the same forward and backwards on the same DNA strand (i.e., single stranded) as in GTAATG. The inverted repeat palindrome is also a sequence that reads the same forward and backwards, but the forward and backward sequences are found in complementary DNA strands (i.e., double stranded) as in GTATAC (Notice that GTATAC is complementary to CATATG). The inverted repeat is more common and has greater biological importance than the mirror-like. Palindromes in DNA sequences 5 ’ 3’ 3’ 5 ’ Genetic palindromes are similar to verbal palindromes. A palindromic sequence in DNA is one in which the 5’ to 3’ base pair sequence is identical on both strands. Restriction endonucleases are categorized into three or four general groups based on their composition and enzyme cofactor requirements. They differ in their recognition sequence, subunit composition, cleavage position, and cofactor requirements: Type I enzymes cleave at sites remote from recognition site; require both ATP and Sadenosyl-L-methionine to function; multifunctional protein with both restriction and methylase activities. Type II enzymes cleave within or at short specific distances from recognition site; most require magnesium; single function (restriction) enzymes independent of methylase. Type III enzymes cleave at sites a short distance from recognition site; require ATP (but doesn't hydrolyse it); S-adenosyl-L-methionine stimulates reaction but is not required; exist as part of a complex with a modification methylase . Type IV enzymes target methylated DNA. Sticky end Sticky end III. Vectors for Gene Cloning A. Requirements of a vector to serve as a carrier molecule The choice of a vector depends on the design of the experimental system and how the cloned gene will be screened or utilized subsequently Most vectors contain a prokaryotic origin of replication allowing maintenance in bacterial cells. Some vectors contain an additional eukaryotic origin of replication allowing autonomous, episomal replication in eukaryotic cells. Multiple unique cloning sites are often included for versatility and easier library construction. Antibiotic resistance genes and/or other selectable markers enable identification of cells that have acquired the vector construct. Some vectors contain inducible or tissue-specific promoters permitting controlled expression of introduced genes in transfected cells or transgenic animals. Modern vectors contain multi-functional elements designed to permit a combination of cloning, DNA sequencing, in vitro mutagenesis and transcription and episomal replication. B. Main types of vectors Plasmid, bacteriophage, cosmid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), retrovirus, baculovirus vector…… C. Choice of vector Depends on nature of protocol or experiment Type of host cell to accommodate rDNA Prokaryotic Eukaryotic Plasmid vectors Plasmid vectors are double-stranded, circular, selfreplicating, extra-chromosomal DNA molecules. Advantages: Small, easy to handle Straightforward selection strategies Useful for cloning small DNA fragments (< 10kbp) Disadvantages: Less useful for cloning large DNA fragments (> 10kbp) A plasmid vector for cloning 1. Contains an origin of replication, allowing for replication independent of host’s genome. 2. Contains Selective markers: Selection of cells containing a plasmid twin antibiotic resistance blue-white screening 3. Contains a multiple cloning site (MCS) 4. Easy to be isolated from the host cell. Plasmid vectors Examples pBR322 One of the original plasmids used Two selectable markers (Amp and Tet resistance) Several unique restriction sites scattered throughout plasmid (some lie within antibiotic resistance genes = means of screening for inserts) ColE1 ORI pUC18 Derivative of pBR322 Advantages over pBR322: Smaller – so can accommodate larger DNA fragments during cloning (510kbp) Higher copy # per cell (500 per cell = 5-10x more than pBR322) Multiple cloning sites clustered in same location = “polylinker” E. Lambda vector Bacteriophage lambda (l) infects E. coli Double-stranded, linear DNA vector – suitable for library construction Can accommodate large segments of foreign DNA Central 1/3 = “stuffer” fragment Can be substituted with any DNA fragment of similar size without affecting ability of lambda to package itself and infect E. coli Accommodates ~15kbp of foreign DNA Foreign DNA is ligated to Left and Right Arms of lambdaThen either: 1) Transfected into E. coli as naked DNA, or 2) Packaged in vitro by combining with phage protein components (heads and tails) (more efficient, but labor intensive and expensive) Left arm: head & tail proteins Right arm: DNA synthesis regulation host lysis Deleted central region: integration & excision regulation F. Cosmid vectors Hybrid molecules containing components of both lambda and plasmid DNA Lambda components: COS sequences (required for in vitro packaging into phage coats) Plasmid DNA components: ORI + Antibiotic resistance gene Cloning sites will be part of vector rDNA is packaged using extracts of coat and tail proteins derived from normal lambda components BUT cannot be packaged after introduced into host cell because rDNA does not encode the genes required for coat proteins After infection of E. coli, rDNA molecules replicate as plasmids Very large inserts can be accommodated by cosmids (up to 35-45 kbp) Disadvantages: Not easy to handle very large plasmids (~ 50 kbp l ZAP G. Shuttle vectors Hybrid molecules designed for use in multiple cell types Multiple ORIs allow replication in both prokaryotic and eukaryotic host cells allowing transfer between different cell types Examples: E. coli yeast cells E. coli human cell lines Selectable markers and cloning sites H. Bacterial artificial chromosomes (BACs) Based on F factor of bacteria (imp. In conjugation) Can accommodate 1 Mb of DNA (= 1000kbp) F factor components for replication and copy # control are present Selectable markers and cloning sites available Other useful features: SP6 and T7 promoters Direct RNA synthesis RNA probes for hybridization experiments RNA for in vitro translation BAC vector oriS and oriE mediate replication parA and parB maintain single copy number ChloramphenicolR marker I. Yeast artificial chromosomes (YACs) Hybrid molecule containing components of yeast, protozoa and bacterial plasmids Yeast: ORI = ARS (autonomously replicating sequence) Selectable markers on each arm (TRP1 and URA3) Yeast centromere Protozoa= Tetrahymena Telomere sequences (yeast telomeres may also be used) Bacterial plasmid Polylinker Can accommodate >1Mb (1000kbp = 106 bp) YAC vector large inserts URA3 HIS3 ARS telomere telomere centromere markers replication origin Capable of carrying inserts of 200 - 2000 kbp in yeast What determines the choice vector? insert size vector size restriction sites copy number cloning efficiency ability to screen for inserts Expression vector J. Human artificial chromosomes Developed in 1997 – synthetic, self-replicating ~1/10 size of normal chromosome Microchromosome that passes to cells during mitosis Contains: ORI Centromere Telomere Protective cap of repeating DNA sequences at ends of chromosome (protects from shortening during mitosis) Histones provided by host cell IV. Constructing Genomic and cDNA Libraries A. Definition A cloned set of rDNA fragments representing either the entire genome of an organism (Genomic library) or the genes transcribed in a particular eukaryotic cell type (cDNA library) rDNA fragments generated using restriction endonucleases rDNA fragments ligated to appropriate cloning vector B. Genomic libraries Commonly bacteriophage lambda used as the vector “Stuffer fragment” removed and replaced with 15-17kbp fragments of library Cosmids and YACs may also be used as vectors Contains at least one copy of all DNA fragments in the complete library Screened using nucleic acid probes to identify specific genes Subcloning is usually necessary for detailed analysis of genes Preparation of genomic library in bacteriophage lambda vector Determination of library size: The larger the fragments that are cloned in a particular vector the smaller the overall size of the library N = ln (1-P)/ ln (1-f) N = Number of required clones P = probability of recovering a desired DNA sequence (P= 0.99) f = fraction of the genome present in each clone (insert) Example: Human genome = 3.2 x 106 kbp = 3.2 x 10 9 bp Lambda vector can accommodate 17kbp inserts N = ln (1 – 0.99) ln [1 – (1.7 x 104 bp insert) 3.2 x 109 bp genome] N = 8.22 x 105 plaques required in library Usually researchers will make genomic libraries 2 – 2.5x the size required using this equation. Human Genome Project (HGP) Entire human genome has been sequenced (April 2000) Project began in 1990 – Joint Venture Human Genome Organization (HuGO) (USA, UK, France, Japan mainly) CELERA This topic will explored in more detail later in the course. C. cDNA libraries mRNA represents genes that are actively transcribed (or expressed) at any given time in a particular cell type Small subsets of sequences found in a genomic library Eukaryotic mRNA = polyadenylated and introns have been removed This is the starting point! mRNA converted into a DNA copy (=cDNA) using a series of enzymatic reactions and oligonucleotides Primer, reverse transcriptase, DNA polymerase I, S1 nuclease, linkers, restriction enzymes, vector Size of library depends on abundance of message Bacteriophage lambda insertion vectors or plasmids are used for cloning The choice depends upon: Abundance of mRNA Size of desired library Screening method Method – cDNA Synthesis and Cloning into a Plasmid Vector 1. mRNA must be separated from other cellular constituents before 1st strand cDNA synthesis is carried out RNA is first purified and DNA is eliminated Isolation of poly(A) RNA using Oligo (dT) cellulose Poly (A) tails of mRNA hybridize to oligo (dT) cellulose resin via column chromatography rRNA and tRNA do not bind and are eluted After extensive washing of the column, then mRNA is eluted by dropping salt concentration, precipitated, washed and quantitated 2. mRNA is combined with an oligo (dT)15-18 synthetic primer which binds to the 3’ end of mRNA 3. Reverse transcriptase is added and synthesis of a DNA copy of the mRNA takes place beginning at 3’ –OH of oligo (dT) primer, extending the cDNA in the 5’ to 3’ direction 4. Alkali treatment degades the mRNA template leaving the first strand of cDNA 5. A hairpin loop forms on the first strand cDNA product. 6. DNA polymerase I is added which extends the hairpin loop back in the 5’ to 3’ direction to complete the second strand cDNA product 7. S1 nuclease digests single stranded ends and the hairpin loop leaving a ds cDNA product with flush ends. 8. Lambda exonuclease is added to nibble back a few nucleotides from the ends to generate short single-stranded overhangs. 9. Terminal deoxynucleotidyl transferase (TdT) is added plus deoxythymidine triphosphate generating strings of Ts at ends of molecules. Alternatively synthetic DNA linkers can be ligated at this stage. 10. cDNA can be cloned into a plasmid with complementary strings of A’s by hydrogen bonding and DNA ligase. If alternative is used above, then the plasmid is digested with appropriate restriction enzyme to produce compatible sticky ends. 11. Recombinant plasmids are transformed into E. coli to produce cDNA library. 12. Screening cDNA libraries is carried out using nucleic acid probes, degenerate oligonucleotide probes, or antibodies. Dependent on resources available and vector used.