Medical Genetics

Recombinant DNA Technology

 Learning outcomes
At the end of the session the student would able to
understand:
• Explain the action and uses of restriction enzymes
• Appreciate the role and various types of Vectors:
• Describe hybridization or annealing of nucleic acids
• Outline the general principles of DNA cloning
• Explain the differences between a cDNA clone and a
genomic clone.
• Genomic Library and cDNA Library.
 Biomedical Importance
• The development of recombinant DNA,
• high-density DNA microarrays,
• high-throughput screening,
• low-cost genome-scale analyses and
• DNA sequencing and other molecular genetic methodologies has
revolutionized biology and is having an increasing impact on
clinical medicine.

• Though much has been learned about human genetic disease

from pedigree analysis and study of affected proteins,
• in many cases where the specific genetic defect is unknown,
these approaches cannot be used.

• The new technologies circumvent limitations by going directly

to the DNA molecule for information.
• Manipulation of a DNA sequence and the construction of chimeric
molecules—so-called genetic engineering—provides a means of
studying how a specific segment of DNA works.

• Novel molecular genetic tools and direct DNA sequencing allow

investigators to query and manipulate genomic sequences as well
as to examine both cellular mRNA and protein profiles at the
molecular level.

• Understanding this technology is important for several reasons:

(1)It offers a rational approach to understanding the molecular basis

of a number of diseases.
For example, familial hypercholesterolemia, sickle cell disease, the
thalassemias, cystic fibrosis, muscular dystrophy as well as more
complex multifactorial diseases like vascular disease, cancer, and
diabetes.
2) Human proteins can be produced in abundance for therapy (eg,
insulin, growth hormone, tissue plasminogen activator).

3) Proteins for vaccines (eg, hepatitis B) and for diagnostic testing

(eg, Ebola and AIDS tests) can be obtained.

4) This technology is used to diagnose existing diseases and predict

the risk of developing a given disease and individual response to
pharmacological therapeutics.

5) Special techniques have led to remarkable advances in forensic

medicine.

6) Gene therapy for potentially curing diseases caused by a single

gene deficiency such as sickle cell disease, the thalassemias,
adenosine deaminase deficiency, and others may be devised.
 What is cloning?
• A clone is an identical copy.
The Human Genome Project

The Human Genome and Individualized

Medicine - David Valle
 DNA Cloning
• DNA cloning involves separating a specific gene or DNA
segment from a larger chromosome,
• attaching it to a small molecule of carrier DNA,
• and then replicating this modified DNA thousands or millions
of times through both an increase in cell number and
• the creation of multiple copies of the cloned DNA in each
cell.

• The result is selective amplification of a particular gene or

DNA segment.
Cloning of DNA from any organism entails five general
procedures:

1. Cutting DNA at precise locations. Sequence-specific endonucleases (restriction

endonucleases) provide the necessary molecular scissors.

2. Selecting a small molecule of DNA capable of self-replication. These DNAs are

called cloning vectors (a vector is a delivery agent). They are typically plasmids or
viral DNAs.

3. Joining two DNA fragments covalently. The enzyme DNA ligase links the cloning
vector and DNA to be cloned. Composite DNA molecules comprising covalently
linked segments from two or more sources are called recombinant DNAs.

4. Moving recombinant DNA from the test tube to a host cell that will provide the
enzymatic machinery for DNA replication.

5. Selecting or identifying host cells that contain recombinant DNA.

• A cloning vector and eukaryotic
chromosomes are separately
cleaved with the same restriction
endonuclease.
• The fragments to be cloned are
then ligated to the cloning vector.
• Bacteria will be treated with
calcium chloride for effective
permeability.
• The resulting recombinant DNA
is introduced(transformation)
into a host cell where it can be
propagated (cloned).
Restriction endonucleases (restriction enzymes)
• Restriction enzymes cut DNA of any source into unique, short
pieces in a sequence-specific manner—in contrast to most
other enzymatic, chemical, or physical methods, which break
DNA randomly.
• Restriction enzymes are named after the bacterium from
which they are isolated.  For example, EcoRI  is from
Escherichia coli.. Etc
• Each enzyme recognizes and cleaves a specific double-stranded DNA
sequence that is typically 4–7 bp long. These DNA cuts result in blunt
ends  (eg, HpaI ) or overlapping (sticky or cohesive) ends (eg, BamHI),
depending on the mechanism used by the enzyme.
• A restriction enzyme that recognizes a 4-bp sequence cuts, on
average, once every 256 bp (44), whereas another enzyme
that recognizes a 6-bp sequence cuts once every 4096 bp (46).
 What is a Palindrome?
• A palindrome is anything that reads the same
forwards and backwards:
• English palindromes: Mom and Dad
• Tarzan raised:Desi Arnaz rat.
• Able was I ere I saw Elba (supposedly said by
Napoleon)
• Doc note I dissent, a fast never prevents a
fatness, I diet on cod.
 DNA Palindromes
• Because DNA is double stranded and the strands
run antiparallel, palindromes are defined as any
double stranded DNA in which reading 5’ to 3’
both are the same
• Some examples:
• The EcoRI cutting site:
– 5'-GAATTC-3'
– 3'-CTTAAG-5'
• The HindIII cutting site:
– 5'-AAGCTT-3'
– 3'-TTCGAA-5'
Restriction Enzymes
 Restriction Enzymes

• Enzymes that cleave double stranded DNA at specific sites

• Cleave at Palindromes

Palindromic recognition
sequence for EcoRI

Creates DNA fragments with sticky ends

 Sticky and Blunt End Restriction Enzymes
• Sticky-end ligation is technically easy, but some special techniques
are often required to overcome problems inherent in this approach.
• Sticky ends of a vector may reconnect with themselves, with no net
gain of DNA.
• Sticky ends of fragments also anneal so that heterogeneous tandem
inserts form.
• Also, sticky-end sites may not be available or in a convenient position.
• To circumvent these problems, an enzyme that generates blunt ends
can be used.
• Blunt ends can be ligated directly, however ligation is not directional.
Cloning
 Vectors (Plasmids) useful in cloning
• Circular DNA molecules
• Contain an origin of replication
• Contain genes for antibiotic resistance
• Have many palindrome sites recognized by
restriction endonucleases
• Other vectors useful in recombinant DNA
technology are: Cosmids, Viral chromosome
(phage), Yeast artificial chromosome (YAC) and
Bacterial artificial chromosome(BAC).
Cloning capacities of common cloning vectors
 PLASMIDS
• Some of the bacteria contain small,
independent circular double
stranded DNA molecules known as
plasmids.
• They provide resistance to the
bacteria against antibiotics..
• Plasmid DNAs replicate
independently and they can be easily
separated from the host bacteria.
• The DNA sequences and restriction
maps (characteristic of endonuclease
action) of many plasmids are known.
Transformation
• This is useful for the incorporation of Electroporation
foreign target DNA into vector. (high voltage
pulse)
• Plamids, the most commonly used
vectors can accept DNA fragments of
6-10 kb length
 Bacteriophages

• These are linear DNA

molecules, containing several
restriction enzyme sites.

• Phages can accept foreign

DNA fragments of 10-20 kb
length

(1kb=1000nucleotide long base

sequence)
 Cosmids
• These are specialized
plasmids containing
DNA sequence
namely Co sites.

• Cosmids can accept

much longer DNA
fragments(20-50 kb)
Multiple Cloning Site
 Properties of a plasmid vector

Plasmids are molecules of DNA found in bacteria.

The basic characteristics of plasmids are:
• circular orientation
• typically only carry a few genes
• small in size (only a few thousand base pairs)
• one origin of replication
 The Role of Plasmids
• The basic procedure in a gene cloning experiment consists of
placing a foreign gene into bacterial cells,
• isolating the individual cells and growing colonies from each
of them.
• All of the cells in each colony are identical to one another
and will contain the foreign gene.
• Therefore as long as the foreign gene can replicate, the gene
can be cloned through the process of cloning its bacterial
host.
• This host serves as a vector, or carrier molecule , for the
gene of interest.
• The result is intermediates with
sticky and complimentary ends.
 
• The foreign gene and plasmid can
then be combined via base-pairing
and linked under incubation with
DNA ligase. 

• As the sticky ends on the vector

and on the gene base-pair
momentarily,
• the ligase acts to seal the nicks,
attaching the two DNAs through
covalent bonds. 

• The foreign gene is thereby

inserted into the plasmid and will
remain in place, unless re-cut with
the same restriction enzyme.
 Plasmids
• Vectors are divided into two groups: phages and plasmids.
• Plasmids frequently serve as carrier molecules in gene cloning
experiments.
• Foreign DNA in such experiments rely on the plasmid as a carrier
molecule for its replication because the gene alone does not
contain an origin of replication. 
• All of the plasmids contain an origin of replication, but that is not
to say that a given gene can be inserted into just any plasmid. 
• The piece of DNA can be inserted as long as both the gene and
the plasmid contain recognition sites conferring for the same
restriction enzymes. 
• The plasmid carrying genes for antibiotic
resistance, and a DNA strand, which
contains the gene of interest, are both cut
with the same restriction enzyme.

• The objective at this point is to grow only

bacteria that solely contain the
recombinant DNA.

• Thus the bacteria can be grown in the

presence of a particular antibiotic for
which the plasmid is resistant. 

• This type of growth selects for cells that

have taken up either the plasmid alone or
the plasmid plus the inserted DNA. 

• Cells that do not receive DNA or received

only inserted DNA will not be resistant to
the antibody;
• these cells should fail to grow. 
• Narrowing the selection process down
even more, the next task is to find the
clones that have received recombinant
DNA.
 Recombinant DNA technology
• The recombinant plasmids are used to transform host cells
(bacteria)
• The host cells with the recombinant vector are selected using
the antibiotic resistance genes
• For example, if the foreign gene is inserted into the tetr gene
→ Inactivation of the tetr gene → The host cell becomes
sensitive to tetracycline but is resistant to ampicillin
 Selecting Colonies

• A common reporter in bacteria is the E. coli lacZ gene, which encodes the protein
beta-galactosidase.

• This enzyme causes bacteria expressing the gene to appear blue when grown on a
medium that contains the substrate analog X-gal.
• Vectors for mammalian cell propagation and insert gene (cDNA)/protein
expression have also been developed.

• These vectors are all based upon various eukaryotic viruses that are
composed of RNA or DNA genomes.

• Notable examples of such viral vectors are those utilizing adenoviral

(Ad), or adenovirus-associated viral (AAV) (DNA-based) and retroviral
(RNA-based) genomes.

• Though somewhat limited in the size of DNA sequences that can be

inserted, such mammalian viral cloning vectors make up for this
shortcoming because they will efficiently infect a wide range of different
cell types.

• For this reason, various mammalian viral vectors are being investigated
for use in gene therapy and are commonly used for laboratory
experiments.
 DNA Libraries
Two types
• Genomic DNA libraries: Contain the
entire nuclear DNA
• cDNA Libraries (Expression libraries):
Contain the cDNA of the mRNA that are
expressed in the tissues
 A Library Is a Collection of Recombinant Clones

• The combination of restriction enzymes and various cloning vectors allows the
entire genome of an organism to be individually packed into a vector.
• A collection of these different recombinant clones is called a library.
• A genomic library  is prepared from the total DNA of a cell line or tissue.
• A cDNA library  comprises complementary DNA copies of the population of
mRNAs in a tissue.
• Genomic DNA libraries are often prepared by performing partial digestion of
total DNA  with a restriction enzyme that cuts DNA frequently (eg, a four base
cutter such as TaqI).
• The idea is to generate rather large fragments so that most genes will be left
intact.
• The BAC, YAC, and P1 vectors are preferred since they can accept very large
fragments of DNA and thus offer a better chance of isolating an intact
eukaryotic mRNA-encoding gene on a single DNA fragment.
• A vector in which the protein coded by the gene introduced by
recombinant DNA technology is actually synthesized is known as
an expression vector.

• Such vectors are now commonly used to detect specific cDNA

molecules in libraries and to produce proteins by genetic
engineering techniques.

• These vectors are specially constructed to contain very active

inducible promoters, proper in-phase translation initiation
codons, both transcription and translation termination signals,
and appropriate protein processing signals, if needed.
 Genomic Library

Genomic library:
Collection of cells that
contain the entire
chromosomal DNA
 Isolate mRNA from cells

Synthesize cDNA using

reverse transcriptase
cDNA
Library
cDNA is inserted into a
plasmid and bacterial cells
are transformed

cDNA library :
Collection of cDNA from
actively transcribed genes
• Construction of a cDNA library
from mRNA.
• A cell’s mRNA includes transcripts from
thousands of genes, and the cDNAs
generated are correspondingly
heterogeneous.

• The duplex DNA produced by this method is

inserted into an appropriate cloning vector.

• Reverse transcriptase can synthesize DNA on

an RNA template.
• A cDNA library can be made
even more specialized by
cloning a cDNA or cDNA
fragment into a vector that
fuses the cDNA sequence
with the sequence for a
marker, or reporter gene;
the fused genes form a
“reporter construct.”

• Two useful markers are the

genes for green fluorescent
protein and epitope tags.
 Comparison of genomic library and cDNA
library
Genomic library
• Chromosomal DNA
• Enzymes required: Restriction
endonuclease, DNA ligase
• Contain introns
• Contain promoter and
enhancer sequences
• Useful for DNA sequencing
(Human genome project)
cDNA library
• cDNA synthesized from mRNA
(only expressed genes)
• Enzymes required: Reverse
transcriptase, DNA polymerase,
DNA ligase
• Contain only exons
• Do not contain promoters and
enhancers
• Useful in producing
recombinant proteins, gene
therapy, constructing transgenic
animals
 Screening of Libraries

Lyse bacteria. Add DNA

probe for the gene and
incubate
Expression
vectors Probe hybridizes to
complementary
sequence on filter
Lyse bacteria. Add labelled
antibodies to bind to protein

Autoradiogram
 Applications of Genomic library
Human Genome project

The genome is cut at specific palindrome sequence using

restriction endonucleases

Restriction fragments are cloned in

vectors

Fragments are re-isolated from the

recombinant vectors using the
restriction endonucleases

Restriction fragments from each clone are

sequenced
 Human Genome Project
Sequencing of the genome is useful for
• Knowing the sequence of the protein coding genes → helps to
identify specific mutations associated with diseases
• Identifying the sites of recognition of restriction endonucleases
(Restriction map)
• Restriction sites can also be used as RFLP markers for genetic
disease
• Genetic markers eg. Tandem repeats, Single nucleotide
polymorphisms (SNP) can also be identified
• Introns (non expressed sequences), regulatory sequences like
enhancers, promoters, response elements can be identified
The Human Genome Project

The Human Genome and Individualized

Medicine - David Valle
 Applications of cDNA cloning
• Production of proteins (recombinant proteins)
• Produce transgenic mice and knockout mice
• Useful to carry out gene therapy in patients
with genetic diseases.
Production of Recombinant proteins

Proteins produced by recombinant DNA

technology are:
• Human Insulin (Diabetes mellitus)
• HBsAg (Vaccine for Hepatitis)
• factor VIII (Hemophilia)
 Gene therapy
• Gene therapy is a technique for correcting defective
genes responsible for disease development.
• A normal gene may be inserted into the genome to
replace a nonfunctional gene. This approach is most
common.
• The gene is not transferred to the offspring
• Vectors useful in gene transfer:
 Retrovirus
 Adenovirus
 Liposome
 Gene therapy
Ex vivo
In vivo gene
gene therapy
therapy
• Modified
Cells fromvector
the patient
is injected
are removed
into patient
and (contains
grown in the
culture
• therapeutic gene)with the modified vector
Cells are infected
• Vector
Gene isinfects cells and
incorporated delivers
into cells the gene into cells
• Cell specific
Modified delivery
cells of gene
are infused is difficult→
back into patientless commonly used
 Reading
• Kaplan, Chapter 6
• Lippincotts 4th Ed, Chapter 33
• Harper’s Biochemistry 26th edition,Chapter 40.