Recombinant DNA Technology

Dr. P. Balaji
Head in Biotechnology
MGR College, Hosur
I. Introduction
A. 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. 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 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
D. Development of
molecular 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 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 (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 Endonucleases
A. 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. Availability
Over 200 enzymes identified, many available commercially from
biotechnology companies
C. 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 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), yeast 2 micron plasmid, retrovirus,
baculovirus vector……
C. Choice of vector
Depends on nature of protocol or experiment
Type of host cell to accommodate rDNA
◦ Prokaryotic
◦ Eukaryotic
D. 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 DNA
Traditional method
Conventional method
Cloning Genes-General
Cloning 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 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 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 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
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)
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.
Cloning ds cDNA in phage vectors
◦ Handout
V. Identification of Specific DNA
Sequences in Libraries
A. 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 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 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 Recovered
by Cloning
A. 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
electrophoresis
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 -
Procedure
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
restriction 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