Recombinant DNA Technology

Sherry Fuller-Espie, Ph.D., DIC

Associate Professor
Cabrini College

© Sherry Fuller-Espie, 2003

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