Recombinant DNA TechnologyRecombinant DNA Technology

Mahesh Mahendrakar

Lecturer in biotechnology

N.G. College

mahendrakar.mahesh@gmail.com

I. IntroductionI. Introduction

A. The Central DogmaA. The Central Dogma

• Gene Expression– DNA makes RNA makes protein

• DNA is transcribed into mRNA (nucleus) which is translated into protein (cytoplasm)

– Transcription of genes relies on the interaction of DNA binding proteins with the regulatory elements of the gene (promoter)• Some genes are activated in all cell types

(housekeeping genes) and are regulated by ubiquitous promoters)

• Some genes are activated in a restricted manner (tissue-specific genes) and are regulated by tissue-specific promoter and enhancer sequences

– Pre-mRNA matures by splicing (the removal of intervening sequences) and polyadenylation (the addition of poly A tracts to the 3’ end), processes believed to stabilize mRNA and assist in its transport to the cytoplasm

– Some mRNA transcripts are differentially spliced giving rise to proteins which may have different functions

– Translation of mature mRNA occurs in the cytoplasm and involves the interaction with tRNA and rRNA complexes

• Codons dictate where translation starts (AUG), stops (UAA, UAG, UGA), and which amino acids to incorporate into the growing polypeptide chain

• Any mutations which have resulted in the addition or removal of nucleotides can alter the reading frame resulting in the translation of non-sense proteins or premature termination of translation.

• Advances in Molecular Biology– The combination of restriction/modification

enzymes and hybridization techniques enable the application of a wide variety of procedures

B. ApplicationsB. 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

C. Definition of C. Definition of recombinant DNArecombinant 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

D. Development of D. Development of molecular biologymolecular biology

• Early research on prokaryotic genetics and the development of molecular techniques has led to a new discipline called MOLECULAR BIOLOGY

• “Tools” have been developed (and still continue to be modified/improved) to enable scientists to examine very specific regions of the genome or genes.

E. Common steps involved in isolating a E. Common steps involved in isolating a particular DNA fragment from a complex particular DNA fragment from a complex mixture of DNA fragments or moleculesmixture 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 (copy #)

• 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 EndonucleasesII. Restriction Endonucleases

A. Origin and functionA. Origin and function

• 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

B. AvailabilityB. Availability

• Over 200 enzymes identified, many available commercially from biotechnology companies

C. ClassesC. Classes

• 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

• 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

• 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 III. Vectors for Gene CloningCloning

A. Requirements of a vector to A. Requirements of a vector to serve as a carrier moleculeserve 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 vectorsB. Main types of vectors

• Plasmid, bacteriophage, cosmid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), yeast 2 micron plasmid, retrovirus, baculovirus vector……

C. Choice of vectorC. Choice of vector

• Depends on nature of protocol or experiment

• Type of host cell to accommodate rDNA– Prokaryotic– Eukaryotic

D. Plasmid vectorD. 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)

• 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 (5-10kbp)

– Higher copy # per cell (500 per cell = 5-10x more than pBR322)

– Multiple cloning sites clustered in same location = “polylinker”

• Interruptable gene encoding for enzyme beta galactosidase (lacZ)

– Polylinker resides in the middle– Enzyme activity can be used as marker for gene

insertion– Disrupted gene = nonfunctional– Intact gene = functional– Media containing XGAL chromagenic substrate used

(blue colonies = intact; white colonies = disrupted)

• Amp resistance gene still present (= beta lactamase), Tet resistance gene omitted

Preparation of plasmid Preparation of plasmid DNADNA

• Traditional method

• Conventional method

Cloning Genes-General Cloning Genes-General Cloning SchemeCloning Scheme

• Vector and foreign gene to be inserted are purified/modified separately before ligating the two together

• Ligated products are introduced into “competent” bacterial cells by transformation techniques. Individual colonies are analyzed separately.

• Vectors able to survive under antibiotic selection are amplified in bacterial hosts by autonomous replication

• Plasmid DNA containing the gene of interest is purified from large scale cultures

• Subsequent steps in the experimental design are undertaken:– Subcloning– Mutagenesis– Sequencing– Transfection of eukaryotic cell lines (calcium phosphate

precipitation, lipofection, electroporation, dextran sulfate, microinjection,…..)

– Fragment isolation for transgenic mice production (microinjection)

– PCR

E. Lambda vectorE. Lambda vector

• Bacteriophage lambda 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 lambda

Then 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)

• Preparation of bacteriophage lambda– Overhead

• Replication cycle of bacteriophage lambda– Overhead

F. Cosmid vectorsF. 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)

G. Shuttle vectorsG. 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 H. Bacterial artificial chromosomes (BACs)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

I. Yeast artificial I. Yeast artificial chromosomes (YACs)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)

J. Human artificial J. Human artificial chromosomes 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 IV. Constructing Genomic and cDNA LibrariescDNA Libraries

A. DefinitionA. 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 librariesB. 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 librariesC. 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 Method – cDNA Synthesis and Cloning into a Plasmid VectorCloning 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.

• Cloning ds cDNA in phage vectors– Handout

V. Identification of Specific DNA V. Identification of Specific DNA Sequences in LibrariesSequences in Libraries

A. Locating specific clonesA. 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

B. Types of probesB. Types of probes

• 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)

– Non-radioactive: Usually based on chemical reactions or color changes

• Chemiluminescence• Colorimetric techniques• Fluorescence (Fluorescence in situ

hybridization = FISH)

• 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)

– 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)

C. Screening librariesC. Screening libraries

• 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

• Expression library– Detect protein product of clone using antibodies

– Microarray technology providing more sophisticated analysis strategies for differential expression of gene products

• 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.

VI. Analysis of DNA VI. Analysis of DNA Recovered by CloningRecovered by Cloning

A. Restriction mappingA. Restriction mapping

• Determination of location and abundance of particular restriction enzyme cutting sites along the length of a DNA fragment

• Information can be useful for subcloning purposes to reduce complexity or for further analysis

• Restriction sites are useful as genetic markers– E.g. if site is close to a mutant gene, the site can

be a useful diagnostic marker

B. Agarose gel B. Agarose gel electrophoresiselectrophoresis

• Separation of DNA fragments based on size, charge and shape differences

• Standardized MW markers run on the same gel for size comparison

• Single and double digests can together aid in the construction of a genetic map

C. Southern blotting - C. Southern blotting - ProcedureProcedure

• Technique developed by Ed Southern used for variety of purposes

• Procedure:– 1. DNA is digested with restriction enzymes

and separated by agarose gel electrophoresis (may be photographed if needed)

– 2. Gel is treated with NaOH to denature DNA ss DNA

– 3. DNA is transferred from gel to a DNA-binding filter (e.g. nitrocellulose or nylon membrane) using capillary action

• Gel sits on a sponge wick. Paper towels absorb rising buffer.

• Buffer passes through the membrane but not the DNA.

• DNA binds to membrane

– 4. DNA is “fixed” by baking membrane at 80oC or UV cross-linking

– 5. The membrane is incubated with ss-nucleic acid probe binds to DNA is complementary. Remainder washed off.

– 6. Autoradiography or chemiluminescence (dep. on probe)

D. Diagnostic markers identified by D. Diagnostic markers identified by restriction mappingrestriction mapping

• Restriction sites close to mutant genes may be different between normal and mutant alleles– Called Restriction Fragment Length

Polymorphisms (RFLPs)– Southern blotting can detect RFLPs by

differences in migration patterns of DNA fragments