This lecture is mainly focused for undergraduate medical (MBBS & BDS) 1st Year students.

1. RECOMBINANT DNA TECHNOLOGY AND
ITS APPLICATIONS
Dr. Apeksha Niraula
Assistant Professor
Department of Biochemistry
BPKIHS

3. Pioneers of Medicine without a
Nobel prize
 Herbert Boyer and Stanley N. Cohen
First Recombinant DNA

4. OBJECTIVES
 Techniques and enzymes used in manipulation of DNA
 DNA Amplification and Cloning
 DNA Library
 Nucleotide Probes
 Polymerase Chain Reaction
 Blotting Techniques
 Applications of Recombinant DNA Technology

5. Introduction
 Recombinant DNA are the altered DNA that results from the insertion of a sequence of
deoxynucleotides not previously present into an existing molecule of DNA by enzymatic
or chemical means
 Recombinant DNA Technology: Gene cloning/ Molecular cloning
 Encompasses a number of experimental protocols leading to the transfer of genetic
information DNA technology (DNA) from one organism to another
 Originally developed as research tools to explore and manipulate genes and to produce the
gene products (proteins)

6.  Any two segments of a single-stranded DNA or RNA, having continuous
complementary sequences of 20 or more bases in common, form complementary
base pairs to form duplex structure
 Duplex may be formed between DNA and DNA, RNA and RNA, or between DNA and
RNA to generate a hybrid
 An application of annealing properties is construction of nucleic acid probes which
provide useful means for detecting genes and mRNAs
Annealing Properties

9. Enzymatic Reactions
Enzymes used in manipualtion of DNA:
I. Restriction Endonucleases
 Bacterial enzymes that cleave specific palindromic sequences in double stranded DNA
 Werner Arber (Nobel Prize 1978)
 Most “Exquisite Scalpels”
 Cleaving the double-stranded DNA very selectively at specific sites, called the
restriction sites
 Each restriction site is a short sequence of 4–6 base pairs, and is palindromic

11.  A restriction enzyme is named according to the organism from which it was isolated
EcoRI
 Escherichia (E) (Genus)
 Coli (co) (Specific)
 Strain Ry13 (R) (Strain)
 First endonuclease (1) (Order of identification)
 An additional letter indicates the type or strain (as needed), and a number (Roman
numeral) is appended to indicate the order in which the enzyme was discovered in that
particular organism
 HaeIII is the third restriction endonuclease isolated from the bacterium
Haemophilus aegyptius

12. Sticky and Blunt Ends
 Restriction enzymes cleave dsDNA so as to produce a 3′-hydroxyl group on one end
and a 5′-phosphate group on the other
 Some restriction endonucleases, such as TaqI produce sticky or cohesive ends
 HaeIII, produce fragments that have blunt ends
 Using the enzyme DNA ligase sticky ends of a DNA fragment of interest can be
covalently joined with other DNA fragments that have sticky ends produced by
cleavage with the same restriction endonuclease

15.  Some restriction enzymes can recognize the sites that are relatively small,
 Four Cutters: Four nucleotides such as Hae III
 Larger sites, Six Cutters: Six nucleotides in the case of Eco RI
 Enzymatic digestion of a DNA molecule by different REs produces several different
fragments of varying sizes depending upon the cutting frequency of the RE
 If a piece of DNA from a species is exposed to a specific RE, a characteristic array of
DNA fragments is produced; this is called RESTRICTION MAP

17. Restriction Site
 A DNA sequence that is recognized and cut by a restriction enzyme is called a
Restriction site
 Restriction endonucleases cleave dsDNA into fragments of different sizes depending
upon the size of the sequence recognized
 For example, an enzyme that recognizes a specific 4-bp sequence produces many cuts in
the DNA molecule, one every 44 bp
 In contrast, an enzyme requiring a unique sequence of 6 bp produces fewer cuts (one
every 46 bp) and, therefore, longer pieces

19. DNA CLONING
 Introduction of a foreign DNA molecule into a replicating cell permits the cloning or,
amplification (that is, the production of many identical copies) of that DNA
 Firstly, the total cellular DNA is first cleaved with a specific restriction enzyme,
creating hundreds of thousands of fragments
 Each of the resulting DNA fragments is joined to a DNA vector molecule (referred to
as a cloning vector) to form a hybrid, or recombinant, DNA molecule
 Each recombinant molecule carries its inserted DNA fragment into a single host cell
(for example, a bacterium), where it is replicated

20.  The process of introducing foreign DNA into a cell is called Transformation for
bacteria and yeast and Transfection for higher eukaryotes
 As the host cell multiplies, it forms a clone in which every bacterium contains copies
of the same inserted DNA fragment, hence the name “Cloning”
 The cloned DNA can be released from its vector by cleavage (using the appropriate
restriction endonuclease) and isolated
 Many identical copies of the DNA of interest can be produced

22.  A vector is a molecule of DNA to which the fragment of DNA to be cloned is joined
Essential properties of a vector:
 Capacity for autonomous replication within a host cell
 Presence of at least one specific nucleotide sequence recognized by a restriction
endonuclease
 Presence of at least one gene (such as an antibiotic resistance gene) that confers the
ability to select for the vector
 Commonly used vectors include plasmids and viruses
Vectors

23.  Prokaryotic organisms typically contain single, large, circular chromosomes
 Most species of bacteria also normally contain small, circular, extrachromosomal DNA
molecules called plasmids
 Plasmids may carry genes that convey antibiotic resistance to the host bacterium and
may facilitate the transfer of genetic information from one bacterium to another
 Can be readily isolated from bacterial cells, their circular DNA cleaved at specific sites by
restriction endonucleases, and up to 15 kb (kilobases) of foreign DNA (cut with the
same restriction enzyme) inserted
Prokaryotic plasmids

25.  Improved vectors that can more efficiently accommodate larger DNA
segments, or express the passenger genes in different cell types
 Bacteriophage λ : Naturally occurring viruses that infect bacteria
 Retroviruses
 Artificial constructs:
 Cosmid
 BAC, YAC, Human artifical chromosomes
 BAC and YAC can accept DNA inserts of 100–300 kb and 250–1,000 kb,
respectively
Other Vectors

26.  A DNA library is a collection of cloned restriction fragments of the DNA of an organism
 Two kinds of libraries are commonly used:
 Genomic libraries
 Complementary DNA (cDNA) libraries
 Genomic libraries: Contain a copy of every DNA nucleotide sequence in the genome
 cDNA libraries: Contain those DNA sequences that only appear as processed
messenger RNA (mRNA) molecules, and these differ according to cell type and
environmental conditions
DNA libraries

27. Gene Cloning

28. POLYMERASE CHAIN REACTION
 PCR is an in vitro method for amplifying a selected DNA sequence that does not rely on
the biologic (in vivo) cloning method
 PCR permits the synthesis of millions of copies of a specific nucleotide sequence in a few
hours
 Can amplify the sequence, even when the targeted sequence makes up less than one part
in a million of the total initial sample
 Can be used to amplify DNA sequences from any source, including viral, bacterial, plant,
or animal

29. Steps: Denaturation Annealing Extension Multiple cycles of the polymerase
chain reaction

30.  A genomic library is created by digestion of the total DNA of an organism with a
restriction endonuclease and subsequent ligation to an appropriate vector
 Recombinant DNA molecules replicate within host bacteria
 Amplified DNA fragments collectively represent the entire genome of the organism and
are called a Genomic Library
Genomic DNA libraries:

31.  cDNA lacks introns and the control regions of the genes
 High level of expression of protein coding gene in a particular tissue allows the
mRNA transcribed from that gene is likely also present at high concentrations in the
cells of that tissue
 For example, reticulocyte mRNA is composed largely of molecules that code for the
α-globin and β-globin chains of hemoglobin A (HbA)
Complementary DNA libraries:

32.  Synthesis of complementary DNA (cDNA)
from messenger RNA (mRNA) using
reverse transcriptase.
 Ligation of double-stranded (ds) DNA
sequences containing a restriction site to
each end allows biologic cloning of cDNA
 **DNA is resistant to alkaline hydrolysis
 dATP, dCTP, dGTP, dTTP =
deoxyadenosine, deoxycytidine,
deoxyguanosine, and deoxythymidine
triphosphates

34. Sequencing cloned DNA fragments
 The base sequence of DNA fragments that have been cloned can be determined
 Sanger dideoxy chain termination method :Original Method
 In this method, the single-stranded DNA (ssDNA) to be sequenced is used as the
template for DNA synthesis by DNA polymerase (DNA pol)
 A radiolabeled primer complementary to the 3′-end of the target DNA is added, along
with the four deoxyribonucleoside triphosphates (dNTP)
 The sample is divided into four reaction tubes, and a small amount of one of the four
dideoxyribonucleoside triphosphates (ddNTP) is added to each tube

37. PROBES
 Cleavage of large DNA molecules by restriction enzymes produces an enormous array
of fragments
 How can the DNA sequence of interest be picked out of such a mixture?
 Answer: Use of a probe, a short piece of ssDNA or RNA, labeled with a radioisotope,
such as 32P, or with a nonradioactive molecule, such as biotin or a fluorescent dye
 Sequence of a probe is complementary to a sequence in the DNA of interest, called the
Target DNA
 Probes are used to identify which band on a gel or which clone in a library contains the
target DNA, a process called screening

39. Allele-specific oligonucleotide (ASO)
probes used to detect the sickle cell
mutation and differentiate between
sickle cell trait and disease

40.  Disposal of radioactive waste is becoming increasingly expensive, nonradiolabeled
probes have been developed
 Most successful: Based on the vitamin biotin, which can be chemically linked to the
nucleotides used to synthesize the probe
 Biotin was chosen: Binds very tenaciously to avidin
 Avidin can be attached to a fluorescent dye detectable optically with great
sensitivity
 Thus, a DNA fragment that hybridizes with the biotinylated probe can be made visible
by immersing the gel in a solution of dye-coupled avidin
Biotinylated probes

41.  After washing away the excess avidin, the DNA fragment that binds the probe is
fluorescent
 Labeled probes can allow detection and localization of DNA or RNA sequences in cell or
tissue preparations, a process called in situ hybridization (ISH)
 If the probe is fluorescent (F), the technique is called FISH

42. Blotting Techniques
 Southern Blotting: Edward Southern
 Combines the use of restriction enzymes, electrophoresis, and DNA probes to
generate, separate, and detect pieces of DNA
 Northern Blotting: For detection of mRNA
 Western Blotting: For detection of Proteins

43. Southern
Blotting

44. Northern Blotting

46.  For understanding the molecular basis of a number of diseases (Familial
hypercholesterolemia, sickle cell disease, thalassemia, cystic fibrosis and muscular dystrophy)
 Human proteins can be produced in abundance for therapy (e.g. Insulin, Growth hormone,
Tissue plasminogen activator)
 Proteins for vaccines (e.g. Hepatitis B) and for diagnostic testing (e.g. AIDS tests) can be
obtained
 Can be used to diagnose existing diseases and predict the risk of developing a given disease
 Gene therapy for Sickle cell disease, Thalassemias, Adenosine deaminase deficiency and
other diseases
Applications

47. AGRICULTURE
ANTIBIOTICS
PRODUCTION
INSULIN
PRODUCTION
DOLION
TRANSGENIC ANIMALS FOR MEAT
TRANSGENIC CHICKEN
APPLICATIONS

48. SUMMARY
 Recombinant DNA Technology
 Restrictions Enzymes: Blunt, Sticky ends, Examples
 Cloning; Probes
 PCR
 DNA Libraries
 Blotting Techniques
 Applications

49. Thank You