RECOMBINANT DNA TECHNOLOGY
- Genetic engineering
- Study how a specific segment of DNA works
- Going directly to the DNA molecule for
information
Why?
- Information explosion
- Offers a rational approach to understanding the
molecular basis of some diseases
- Human proteins can be produced in abundance
for treatment, vaccines and diagnostic tests
- Diagnosis of existing disease
- Predicting the risk of developing a given disease
- Forensic medicine
- Gene therapy
Recombinant DNA technology Basis
- Elucidation of the basic features of DNA
DNA
- A complex biopolymer
- Organized as a double helix
- Fundamental organizational element sequence
of purine and pyrimidine bases
- Bases: attached to the C-1’ portion of the sugar
deoxyribose
- Bases: linked together through joining of the
sugar moieties at their 3’ to 5’ position via
phosphodiester bond
- Backbone of the double helix : alternating
deoxyribose and phosphate groups
- 3’-5’ linkages define orientation of a given
strand
- 2 strands run in opposite direction:
ANTIPARALLEL
- Base pairing – fundamental
- Base pairs: Complementary
o G- C
o T-A
- Held by: base pairing and hydrophobic base-
stacking interactions
- reduced by heating: DENATURATION
- Degree of base-pair matching (or mismatching)
– estimated from the temperature required for
denaturation – renaturation
- A closely matched segment will withstand more
heat before the strands separate
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- HYBRIDIZATION
- 3 x 10*8 base pairs in each human haploid gene
- Double-helical DNA is packaged into a more
compact structure by a number of proteins-
basic proteins (HISTONES)
- 1m long few cubic microns
Prokaryotic gene
- Small regulatory region (100-500 bp)
- Large protein – coding segment ( 500-10,000bp)
- Control : single regulatory unit
- Coding regions are interrupted by non-coding
regions
Exons
- Regions that appear in mature RNA spp
- Coding regions
INTRONS/non coding
- Interpose or intervene between exons always
removed from precursor RNA before transport
into the cytoplasm occurs
- Process: RNA splicing
- Incorrect processing of primary transcription
 Mature mRNA ::: disease
 More of the DNA gene are introns
 Introns are larger
 Regulatory regions for specific eukaryotic genes
– located in the DNA that flanks the
transcription initiation site at the 5’ end
 5’ flanking- sequence DNA
Mammalian cell
- each gene has its own regulatory region
- complicated multicomponent structure
- information generally flows form DNA to mRNA
to protein
- rigidly controlled process
Enhancers
- special regions that increase the rate of
transcription
Silencers
- repress transcription
Recombinant DNA
- Altering the genetic makeup of an organism by :
1. Adding new DNA to it
2. Changing the DNA that is already there

Recombinant DNA technology

- Isolation and manipulation of DNA molecule to
make chimeric molecules
- Includes end-to-end joining of sequences form
very different sources
- Essence of research
- Several unique techniques and reagents

RECOMBINANT DNA TECHNOLOGY

- Key tools: restriction enzymes

Endonucleases
- Enzyme that cut DNA at specific DNA sequence
within the molecule
Exonucleases
- Digest form the ends of DNA molecule

Restriction enzymes
Types of DNA preparation:
1. COPY OR COMPELEMENT DNA (cDNA)
- Generated from mRNA in a given tissue
- Enzyme : reverse transcriptase
2. GENOMIC DNA
- Digest extracted DNA with a restriction enzyme
- Cut DNA of any source into short pieces in a
sequence-specific manner
- Protect the short bacterial DNA form DNA from
foreign organisms (infective phages)
- Only present in cells that also have a companion
enzyme that methylates the host DNA
- Site-specific DNA methylases and restriction
enzymes always exist in pairs in a bacterium
- Named after the bacteria from which they are
isolated
- Eco RI
- Each enzyme recognizes and cleaves a specific
double-stranded DNA sequence (4-7 bp long)
- Result: sticky end
Blurt ends

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- Sticky ends : useful in constructing hybrid or
chimeric molecules
- Restriction Map : given a piece of DNA 
characteristic linear array of sites for various
enzymes
- DNA digested  ends of all the fragments have
the same DNA sequence
- Fragments produced isolated by electrophoresis
on agarose or polyacrylamide (essential step in
cloning and major use of this enzymes)
- STICKY ENDS OF A VECTOR
Possible problems:
 May reconnect with themselves  no
net gain of DNA
 Can anneal  heterogenous inserts
form
 Sites may not be available
 Inconvenient position
Solution:
o An enzyme that generates blunt ends is
used  new ends are added using
terminal transferase
o Product : homopolymer tailing
DNA-dependent POLYMERASES
- Make complementary copies of DNA templates
 Production of labeled probes:
- 32P- 35S –containing nucleotides to ensure
detection
- Probe must recognize a complementary
sequence to be effective
 DNA probes
- Used to detect DNA on southern blot and
detect and quantitate RNA on northern blot
 DNA sequencing
 DNA amplification
- PCR – automated method of amplifying
segment of DNA
DNA LIGASE
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- Catalyzes that formation of a phosphodiester
bond between adjacent 3’ - hydroxyl and 5’ –
phosphate ends of DNA
- Essential in forming recombinant DNA clones
RNA-dependent DNA POLYMERASES
- Derived from RNA tumor viruses that make
cDNA copies of RNA templates
- Useful in cloning sequences that are
complementary to Mrna
VECTORS
- Restrictions digest containing the target DNA is
recombined in a specific location within a carrier
(vector)
- Essential features:
 Able to replicate
 Capable of insertion
 Selectable marker
 Contain a site for insertion of target
DNA
Types of vectors:
1. Plasmids
- Extrachromosomal circular pieces of DNA
found in bacteria
- Can replicate within the host bacterium
- Carry genes that code for proteins that confer
antibiotic resistance to the host
 2. Bacteriophages
- Viruses that infect the bacteria that had been
genetically modified to produce cloning
vectors
- Advantage of phage over plasmid – more
efficient in transfecting the host
- Can hold bigger fragment of DNA
3. Cosmid
- Plasmids that contain DNA sequences
(cossites) required for packaging lambda DNA
into package particle
- Grow in the plasmid form in bacteria
- Will accept and propagate DNA inserts 35 to
50 KB long
4. YAC (yeast artificial chromosome)
- Presence of introns in human genes requires
vectors that accept much bigger pieces of
DNA
- Accept 1000 KB inserts and are easily
propagated in yeast cells
- Helpful in studying the human genome
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5. Clone
- Cells within an identical genotype
- Identical host cells that carry an identical
recombinant DNA molecule
- A large population of identical molecules,
bacteria or cells that arise form a common
ancestor
- Allow for the production of a large number of
identical DNA molecules, which can be
characterized or used for other purposes
BASIC CLONING STRATEGY
- A recombinant DNA is made from vector and
target DNA by annealing and ligation
- Recombinant DNA molecule is introduced into
the host : TRANSFORMATION or TRANSFECTION
- Propagation in the host cells
- Target DNA is specifically selected by
hybridization
GENE CLONING STEPS:
1. Vector and insert DNA are digested with the
same restriction enzyme  ends of insert DNA
and vector DNA match  reanneal
2. Restriction digests of vector and insert DNA are
mixed and allowed to reanneal
3. After the DNA strands are sealed with DNA
ligase  Vector is propagated to host bacteria
(genetically engineered to facilitate
propagation)
4. Target DNA selected by various detection
procedure
 - Detects RNA by using labeled polynucleotides
with complementary sequences to the target
RNA
- To detect and quantitate mRNA’s from
different tissues
- Allows one to observe a particular gene’s
expression pattern between tissues, organs,
developmental stages, environmental stress
levels, pathogen infection, and over the
course of treatment
- Show overexpression of oncogenes and
downregulation of tumor-suppressor genes in
cancerous cells when compared to ‘normal’
tissue
- Gene expression in the rejection of
MAJOR ANALYTIC TECHNIQUES transplanted organs

1. Southern Blot
- Verify the presence or absence of a specific
nucleotide sequence in the DNA form
different sources
- Gene discovery and mapping
- Evolution and development studies
- Diagnostic and forensics
- Genetically modified organisms → definitive
test to ensure that a particular section of DNA
of known genetic sequence has been
successfully incorporated into the genome of
the host organism
- Detects DNA by using labeled polynucleotides
with complementary sequences to the target
DNA fragment
- Fragments is generated by restriction enzyme
digestion within a mixture of all of the
restriction enzyme fragments of a genome 3. Western Blot
- Detects proteins by using labeled antibodies
to the proteins
- Determine in which tissues, or under which
physiological conditions a gene is expressed to
produce a protein
- Determine whether a transgenic organism
expresses the inserted gene to produce a
protein

- PRENATAL DIAGNOSIS
 10 ml of amniotic fluid
 Chorionic villi biopsy
 Restriction pattern AA for sickle cell
disease

2. Northern Blot

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 4. Restriction Fragment Length Polymorphism 6. Polymerase chain reaction
(RFLP) - Repetitive rounds of DNA synthesis between
- Restriction fragments of different sizes are two primers are used to amplify DNA
obtained on tx with restriction endonucleases  For Dx
- Polymorphisms are analyzed using gel  Forensic med
electrophoresis to separate fragments by size  Tissue typing
→ blotting → annealing of a probe for a gene
being sought

5. DNA sequencing
- DNA can be sequenced either
chemically(Maxam and Gilbert) or
enzymatically (Sanger)
- Sanger procedure is easier and qualitatively
superior that the chemical procedure

Part of a radioactively
labelled sequencing gel

 DNA testing is currently the most advanced and

Sequence ladder by
radioactive sequencing
compared to fluorescent
peaks
accurate technology to determine parentage
 In a DNA parentage test, the probability of
parentage is 0% when the alleged parent is not
biologically related to the child and the
probability of paternity typically greater that
99.9% when the alleged parent is biologically
related to the child
 The current techniques for paternal testing are
using POLYMERASE CHAIN REACTION (PCR) and
RESTRICTION FRAGMENT LENGTH
POLYMORPHISM

7. DNA fingerprinting
- Variation of RLFP analysis
- Based on repetitive sequences that is highly
polymorphic

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 - Attempts to prove genetic identity by
comparing RFLP’s form unknown sample with
a known sample
- Basis of fingerprints
IMPORTANCE RESEARCH & DIAGNOSIS
 Production of materials for biomedical
application
 Can supply large amount s of material that
cannot be obtained by conventional purification
methods
 Plasminogen activation factors
 Interferon

 Provide human material

 Insulin
 Growth hormone
 Aim to supply products (proteins)
 Treatment : insulin
 Diagnosis : AIDS test
 Prevention : Vaccines

8. Gene therapy
- Disease caused by deficiency of a gene
product are amenable replacement therapy

9. Transgenic animals
- Germ line modification devised and tested
only in experimental animals

 Cell-cell differences are determined by

expression of different sets of genes
 The secret lies in the CHROMATIN

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