Recombinant DNA Technology_2004_NO_FIGS

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Recombinant DNA Technology : 

Recombinant DNA Technology Sherry Fuller-Espie, Ph.D., DIC Associate Professor Cabrini College © Sherry Fuller-Espie, 2004

Definition of Recombinant DNA : 

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

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Advances in Molecular Biology The combination of restriction and modification enzymes combined with hybridization techniques enable the application of a wide variety of procedures

Applications : 

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

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Recombinant Vaccines Genetically modified crops Biosensors Monoclonal antibodies Cell/tissue culture Xenotransplantation Bioremediation Production of next generation antibiotics Forensics Bioterrorism detection

Isolating a Particular Gene of Interest : 

Isolating a Particular Gene of Interest 1. DNA molecules are digested with enzymes called restriction endonucleases which reduces the size of the fragments Renders them more manageable for cloning purposes

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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 (copy #)

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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 per cell after DNA replication As host cell divides, rDNA partitioned into daughter cells

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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 : 

II. Restriction Endonucleases

Origin and function of Restriction Endonucleases : 

Origin and function of Restriction Endonucleases Bacterial origin = enzymes that cleave foreign DNA Named after the organism from which they were derived EcoRI from Escherichia coli BamHI from Bacillus amyloliquefaciens Protect bacteria from bacteriophage infection Restricts viral replication Bacterium protects it’s own DNA by methylating those specific sequence motifs

Availability of Restriction Endonucleases : 

Availability of Restriction Endonucleases Over 2500 enzymes have been identified, recognizing ~200 distinct sequences 4 – 8 bases in length. Many are available commercially from biotechnology companies

Two Classes of Restriction Endonucleases: Type I and Type II : 

Two Classes of Restriction Endonucleases: Type I and Type II Type I Cuts the DNA on both strands but at a non-specific location at varying distances from the particular sequence that is recognized by the restriction enzyme Therefore random/imprecise cuts Not very useful for rDNA applications

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Type II Cuts both strands of DNA within the particular sequence recognized by the restriction enzyme Used widely for molecular biology procedures DNA sequence = symmetrical

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Reads the same in the 5’ 3’ direction on both strands = Palindromic Sequence Some enzymes generate “blunt ends” (cut in middle) Others generate “sticky ends” (staggered cuts) H-bonding possible with complementary tails DNA ligase covalently links the two fragments together by forming phosphodiester bonds of the phosphate-sugar backbones

III. Vectors for Gene Cloning : 

III. Vectors for Gene Cloning

Requirements of a vector to serve as a carrier molecule : 

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 and propagation in bacterial cells.

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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.

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Antibiotic resistance genes and/or other selectable markers enable identification of cells that have acquired the vector construct. Antibiotic resistant cells survive = transformed Antibiotic sensitive cells die = untransformed Some vectors contain inducible or tissue-specific promoters permitting controlled expression of introduced genes in transfected cells or transgenic animals.

Many types of vectors to choose from : 

Many types of vectors to choose from Plasmid, bacteriophage, cosmid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), yeast 2 micron plasmid, retrovirus, baculovirus vector……

Choice of vector : 

Choice of vector Depends on nature of protocol or experiment Type of host cell to accommodate rDNA Prokaryotic Eukaryotic

Example: Plasmid vector : 

Example: Plasmid vector Covalently closed, circular, double stranded DNA molecules that occur naturally and replicate extrachromosomally in bacteria Many confer drug resistance to bacterial strains Origin of replication present (ORI)

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Example: pUC18 Small, 2.7kb – so can accommodate relatively large DNA fragments during cloning (5-10kbp) High copy # per cell (500 per cell) – pUC origin or replication Multiple cloning sites clustered in same location = “polylinker” Ampicillin resistance marker

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Interruptable gene encoding enzyme b galactosidase (lacZ) Polylinker resides in the middle of lacZ Enzyme activity can be used as marker for gene insertion Disrupted gene = nonfunctional Intact gene = functional Lac operon is induced with IPTG (in media) IPTG = isopropylthio-b-D-galactoside Media also contains XGAL chromagenic substrate used (blue colonies = intact; white colonies = disrupted) XGAL = 5-bromo-4-chloro-3indoyl-b-D-galactoside

Cloning Genes in Plasmids : 

Cloning Genes in Plasmids Vector and foreign gene to be inserted are purified/modified separately before ligating the two together (DNA ligase) Ligated products are introduced into “competent” bacterial cells by transformation techniques Calcium chloride Electroporation

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Selection is applied Only those cells expressing selectable marker survive (e.g. antibiotic resistance) Colonies are screened Colony hybridization using complementary DNA probes Plasmid DNA containing the gene of interest is purified from selected colony for analysis

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What next? Subcloning into an alternative vector Mutagenesis to study effects of nucleotide substitutions Sequencing to determine DNA sequence Introduction into eukaryotic cell lines to analyze function of the gene product (transfection using calcium phosphate precipitation, lipofection, electroporation, dextran sulfate, microinjection,…..) Production of transgenic mice

IV. Constructing Genomic and cDNA Libraries : 

IV. Constructing Genomic and cDNA Libraries

A. Definition : 

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 : 

B. Genomic libraries Vector choice varies Bacteriophage lambda, Cosmids, YACs, BACs…. Contains at least one copy of all DNA fragments in the complete library Screened using suitable probe to identify specific genes or gene products Subcloning is usually necessary for detailed analysis of genes

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Example: Preparation of genomic library in bacteriophage lambda vector Cloning vector prepared Source DNA prepared Ligation of source DNA with vector In vitro packaging Infection of E. coli Generation of lawn of bacteria containing plaques Each plaque represents a single clone containing one recombinant piece of DNA inserted into the vector

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Determination of library size: The larger the fragments that are cloned in a particular vector  the smaller the overall size of the library

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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)

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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.

C. cDNA libraries : 

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!

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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

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Bacteriophage lambda insertion vectors or plasmids are often used for cloning cDNA libraries The choice depends upon: Abundance of mRNA Size of desired library Screening method

Method – cDNA Synthesis and Cloning into a Plasmid Vector : 

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

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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

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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

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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.

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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.

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11. Recombinant plasmids are transformed into E. coli to produce the 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.

V. Identification of Specific DNA Sequences in Libraries : 

V. Identification of Specific DNA Sequences in Libraries Finding a needle in a haystack

Locating specific clones : 

Locating specific clones Libraries must be “searched” using a specific probe Specificity is important to eliminate irrelevant background Only genes of interest, or those closely related, should be identified in the screening process

Types of probes for screening libraries : 

Types of probes for screening libraries Most probes are single-stranded nucleic acid fragments complementary to the gene being sought Radioactive versus non-radioactive alternatives Radioactive: Radioisotopes serve as the tag for identifiying where the probe has bound to desired genomic or cDNA clones Autoradiography required (X-ray film exposed to radioactivity)

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Non-radioactive: Usually based on chemical reactions or color changes Chemiluminescence Colorimetric techniques Fluorescence (Fluorescence in situ hybridization = FISH)

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Sources of probes Heterologous probes From another species (provided genes are highly conserved) “Phone-a-clone” cDNA probes To recover genomic sequences when introns and promoter elements are needed Probe based on protein sequence If the amino acid composition of a protein is known, then degenerate oligonucleotide probes can be generated 18-21 bases is sufficient for specific probe (6-7 aa)

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Oligonucleotide probes Short synthetic ssDNA RNA probes Generated via in vitro transcription with RNA polymerase from SP6 or T7 promoter Antibodies Used for “expression” libraries (lambda gt11) Fusion proteins (beta galactosidase + cDNA product)

Screening Libraries with Probes : 

Screening Libraries with Probes Plasmid library Bacterial colonies Bacteriophage library Plaques (much smaller, more can be screened per plate!) Method is the same Replica of colonies/plaques transferred to filters Filter treated with solutions that will lyse the bacterial cell walls and denature the DNA (ds ss) Heating/Drying to bind ssDNA to filter permanently Probing (binds if complementary) Wash off unbound probe Autoradiography/Appropriate detection system

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Expression library Detect protein product of clone using antibodies Chromosome walking If nearby sequences have been cloned, this can be used as a starting point for isolation of adjacent genes Contiguous chromosomal sequences used as probes for each round of screening.

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