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 its 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 nonspecific 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 tissuespecific 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 (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 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, selfreplicating
~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 1517kbp 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 As 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 DNAbinding 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 ssnucleic 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