Recombinant DNA technology involves manipulating DNA sequences in the laboratory. DNA is isolated, cut with restriction enzymes, and joined with DNA ligase. The recombinant DNA is inserted into a cloning vector and introduced into a host cell. Cells containing the recombinant DNA are selected and amplified. This allows large quantities of identical DNA molecules to be produced for analysis, comparison, and other purposes. Common applications include understanding disease, producing therapeutic proteins, disease prevention through vaccines, diagnosis, and gene therapy.

1. Recombinant
DNA Technology
part 1
NAMRATA CHHABRA

2. What is Recombinant DNA ?
● Recombinant DNA, also known as
Chimeric DNA
● Contains sequences taken from
very different sources,
● e.g. DNA containing both human
and bacterial DNA sequences
Pig-monkey hybrids are born alive in China

3. Recombinant DNA Technology
● Laboratory manipulation of the inherited characteristics of cells and
organisms.
The technology involves:
● Excising
● Joining, and
● Cloning specific sequences of DNA

4. Genetic engineering
Manipulation of a DNA sequence
and the construction of chimeric
molecule is also called genetic
engineering.

5. Purpose of Recombinant DNA technology
This is the laboratory manipulation of
nucleic acids to-
• Analyze
• Compare
• Construct
• Mutate

6. Purpose of genetic engineering
(1) Understanding the molecular basis of diseases.
For example,
● familial hypercholesterolemia,
● sickle-cell disease,
● thalassemias,
● cystic fibrosis,
● muscular dystrophy,
● vascular and heart disease,
● Alzheimer disease, cancer, obesity and diabetes.

7. Purpose of genetic engineering
(2) Synthesis of human proteins in abundance for therapeutic
purpose eg,
● Insulin,
● Growth hormone,
● Tissue plasminogen activator
● Vaccines etc.

8. Purpose of genetic engineering
(3) Disease prevention -vaccines (eg, hepatitis B)
(4) Diagnosis- for diagnostic testing (eg, Ebola and AIDS tests)
(4) Risk prediction of developing a given disease and
(5) Prediction of response to pharmacological therapeutics.

9. Purpose of genetic engineering
(5) Crime detection- DNA fingerprinting, 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.

10. Steps involved in Recombinant DNA Technology
The procedure involves:
1) Isolation of DNA
2) Cutting DNA at precise locations
3) Joining two DNA fragments covalently
4) Selection of a small molecule of DNA capable of self- replication
5) Moving recombinant molecules from the test tube in to a host cell
6) Selecting or identifying those cells that contain Recombinant DNA

11. 1) Isolation of DNA
● The first step involves isolating the desired
DNA in its pure form i.e. free from other
macromolecules.
● Since DNA exists within the cell membrane
along with other macromolecules such as
RNA, polysaccharides, proteins, and lipids, it
must be separated and purified.

12. 2) Cleavage of DNA
● Special enzymes, Restriction enzymes cut DNA
chains at specific locations.
● These enzymes are also called “Molecular
scissors”
● The specific site is called “Restriction site” which
is 4-7 base pair long
● The fragments of DNA obtained after the action of
restriction enzymes are called “Restriction
fragments”; they can have sticky or blunt ends.
●

13. Restriction endonucleases
● These enzymes were called restriction enzymes because their
presence in a given bacterium restricted (ie, prevented) the growth of
certain bacterial viruses called bacteriophages.
● Endonucleases—enzymes that cut DNA at specific DNA sequences
within the molecule (as opposed to exonucleases,which processively
digest from the ends of DNA molecules)

14. Restriction endonucleases
How is the host DNA protected?
Restriction endonucleases are present only in cells that also have a
companion enzyme that site-specifically methylates the host DNA,
rendering it an unsuitable substrate for digestion by that particular
restriction enzyme.
Thus, site-specific DNA methylases and restriction enzymes that
target the exact same sites always exist in pairs in a bacterium.

15. Site specific cleavage
● Each enzyme recognizes and cleaves a specific double-stranded DNA
sequence that is typically 4 to 7 bp long (restriction site).
● Most recognition sequences are palindromes (ie, the sequence reads the
same in opposite directions on the two strands).
● By convention, these are written in the 5′ to 3′ direction for the upper strand of
each recognition sequence, and the lower strand is shown with the opposite
(ie, 3′-5′) polarity.

16. Nomenclature of Restriction endonucleases
● Restriction enzymes are named after the bacterium from which they
are isolated.
● For example, EcoRI is from Escherichia coli, and BamHI is from
Bacillus amyloliquefaciens.
● The first three letters in the restriction enzyme name consist of the
first letter of the genus (E) and the first two letters of the species (co).
● These may be followed by a strain designation (R) and a roman
numeral (I) to indicate the order of discovery (eg, EcoRI and EcoRII)

17. Restriction fragments
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.

18. Sticky ends
● Sticky, or complementary cohesive-end ligation of DNA fragments is
technically easy.
● They are particularly useful in constructing hybrid or chimeric DNA molecules
● However, 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

20. Blunt ends
To circumvent these problems, an enzyme that generates blunt ends can be used.
Blunt ends can be ligated directly; however, ligation is not directional.
Two alternatives thus exist:
Homopolymer tailing
New ends are added using the enzyme terminal transferase or synthetic sticky
ends are added. If poly d(G) is added to the 3′ ends of the vector and poly d(C) is
added to the 3′ ends of the foreign DNA using terminal transferase, the two
molecules can only anneal to each other, thus circumventing the problems listed
above.

21. Blunt ends
Alternatively, synthetic blunt-ended duplex oligonucleotide linkers containing the
recognition sequence for a convenient restriction enzyme sequence are ligated to
the blunt-ended DNA.
Direct blunt-end ligation is accomplished using the bacteriophage T4 enzyme DNA
ligase. This technique, though less efficient than sticky-end ligation, has the
advantage of joining together any pairs of ends.

23. A revision of mechanism of action of restriction endonuclease

24. 2) Joining two DNA fragments covalently
The DNA fragments are joined
together by DNA ligase

25. DNA Ligase
In DNA replication, ligase’s function is to join together fragments of newly
synthesized DNA to form a seamless strand.
The ligases used in DNA cloning do basically the same thing.
If two pieces of DNA have matching ends, DNA ligase can join them together to
make an unbroken molecule.

26. DNA Ligase
● Using ATP as an energy source, ligase catalyzes a reaction in which the
phosphate group sticking off the 5’ end of one DNA strand is linked to the
hydroxyl group sticking off the 3’ end of the other.
● This reaction produces an intact sugar-phosphate backbone.

27. Example- Use of restriction endonuclease and ligase

28. Example- Use of restriction endonuclease and ligase

29. Example- Use of restriction endonuclease and ligase

30. Recombinant Plasmid

31. III) Cloning
A clone is a large population of identical
molecules, bacteria, or cells that arise from a
common ancestor.
Molecular cloning allows for the production
of a large number of identical DNA
molecules, which can then be characterized
or used for other purposes.

32. Cloning vectors
● Chimeric or hybrid DNA molecules can be inserted in cloning vectors which
then continue to replicate in a host cell under their own control systems.
● In this way, the chimeric DNA is amplified.

33. Cloning vectors
A vector is a molecule of DNA to which the fragment of DNA to be cloned is
attached.
Commonly used vectors are:
• Plasmids
• Bacterial and animal viruses
• Cosmids
• Artificial chromosomes

34. Cloning vectors

35. Bacterial Plasmids
● Bacterial plasmids are small, circular, duplex DNA molecules whose natural
function is to confer antibiotic resistance to the host cell.
● They exist as single or multiple copies within the bacterium and replicate
independently from the bacterial DNA
● Plasmids have several properties that make them extremely useful as cloning
vectors.

36. Plasmids
1. Origin of replication
2. Site for restriction endonucleases
3. Identification markers (antibiotic
resistance genes)

37. Plasmids
● Plasmids range in size from a few thousand base pairs to more than 100
kilobases (kb).
● Like the host-cell chromosomal DNA, plasmid DNA is duplicated before every
cell division.
● During cell division, at least one copy of the plasmid DNA is segregated to
each daughter cell, assuring continued propagation of the plasmid through
successive generations of the host cell.

38. Plasmids as cloning vectors
● The plasmids most commonly used in recombinant DNA technology replicate
in E. coli.
● Generally, these plasmids have been engineered to optimize their use as
vectors in DNA cloning.
● For instance, to simplify working with plasmids, their length is reduced; many
plasmid vectors are only ≈3kb in length, which is much shorter than in
naturally occurring E. coli plasmids.

39. Bacterial and animal viruses
● Phages usually have linear DNA molecules
into which foreign DNA can be inserted at
several restriction enzyme sites.
● A major advantage of phage vectors is that
while plasmids accept DNA pieces about 6–
10 kb long, phages can accept longer DNA
fragments.

40. Bacteriophage- a cloning vector

41. Cosmids as cloning vectors
● Larger fragments of DNA can be cloned in cosmids
● Combine the best features of plasmids and phages.
● Contain the DNA sequences, so-called cos sites,
required for packaging lambda DNA into the phage
particle.
● These vectors grow in the plasmid form in bacteria,
● Chimeric DNA can be packaged into the particle
head.
● Cosmids can carry inserts of chimeric DNA that are
35 to 50 kb long.

42. Cosmids

43. Artificial Chromosomes
Even larger pieces of DNA can be incorporated
into bacterial artificial chromosome (BAC) or
yeast artificial chromosome (YAC).
These vectors can accept and propagate DNA
inserts of several hundred kilobases or more and
have largely replaced the plasmid, phage, and
cosmid vectors.

44. Yeast artificial chromosome
YACs contain selection, replication,
and segregation functions that work in both bacteria and yeast cells and therefore can be propagated in
either organism.

45. Viral vectors
Mammalian viral vectors are also
used in this technology especially
for gene therapy.

46. 4) Moving recombinant molecules from the test tube into a host
cell
E. coli is the most common host cell due to the
following advantages:
i) It’s DNA metabolism is well understood
ii) Cloning vectors associated with E.Coli are well
characterized
iii)Effective techniques are available for moving DNA
from one bacterial cell to another

47. Transformation
● The term transformation is used to denote the genetic alteration of a cell
caused by the uptake and expression of foreign DNA regardless of the
mechanism involved.
● The phenomenon of transformation permits plasmid vectors to be introduced
into and expressed by E. coli cells.
● Normal E. coli cells cannot take up plasmid DNA from the medium.
● Exposure of cells to high concentrations of certain divalent cations, however,
makes a small fraction of cells permeable to foreign DNA by a mechanism
that is not understood.

48. Transformation
● In a typical procedure, E. coli cells are
treated with CaCl2 and mixed with plasmid
vectors;
● commonly, only 1 cell in about 10,000 or
more cells becomes competent to take up
the foreign DNA.
● Transformation can also be undertaken by
giving a heat shock

49. 5) Selecting or identifying those cells that contain Recombinant
DNA
● Colony or plaque hybridization is the
method by which specific clones are
identified and purified.
● Bacteria are grown as colonies on an
agar plate.

50. Colony or plaque hybridization
Colonies are overlaid with
nitrocellulose filter paper.
Cells from each colony stick to the
filter and are permanently fixed
thereto by heat, which with NaOH
treatment also lyses the cells and
denatures the DNA so that it will
hybridize with the probe.
https://www.youtube.com/watch?
v=PEwWXrRWvWE

51. Selecting or identifying those cells that contain Recombinant DNA
● A radioactive probe is added to the filter, and (after
washing) the hybrid complex is localized by
exposing the filter to x-ray film.
● By matching the spot on the autoradiograph to a
colony, the latter can be picked from the plate.
● A similar strategy is used to identify fragments in
phage libraries.
● Successive rounds of this procedure result in a
clonal isolate (bacterial colony) or individual phage
plaque.

52. Colony or plaque hybridization

53. Viral vectors
● Adenoviral (Ad),
● 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.

54. Gene therapy

55. DNA library
● Each colony is a cell clone, but it is also a DNA clone because the
recombinant vector has now been amplified by replication during every round
of cell division.
● Thus, the Petri dish, which may contain many hundreds of distinct colonies,
represents a large number of clones of different DNA fragments.
● This collection of clones is called a DNA library.

56. Genomic library
● A genomic library is prepared from the total DNA of a cell line or 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

57. Genomic library
By considering the size of the donor
genome and the average size of the
inserts in the recombinant DNA molecule,
a researcher can calculate the number of
clones needed to encompass the entire
donor genome, or, in other words, the
number of clones needed to constitute a
genomic library.

58. cDNA library
● Creation of a cDNA library begins with messenger ribonucleic acid (mRNA)
instead of DNA.
● Messenger RNA carries encoded information from DNA to ribosomes for
translation into protein.
● To create a cDNA library, these mRNA molecules are treated with the
enzyme reverse transcriptase, which is used to make a DNA copy of an
mRNA.
● The resulting DNA molecules are called complementary DNA (cDNA).
● A cDNA library represents a sampling of the transcribed genes, whereas a
genomic library includes untranscribed regions.

59. cDNA library
A cDNA library comprises
complementary DNA copies of the
population of mRNAs in a tissue.
It represents a collection of only the
genes that are encoded into proteins by
an organism.

60. Expression vectors
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.
● Some expression vectors even contain
genes that code for protease inhibitors, so
that the final yield of product is enhanced.

61. DNA Probes
https://www.youtube.com/watch?v=
3LtoKv-XjCo

62. Blot transfer
Visualization of a specific DNA or RNA fragment among the many thousands of
“contaminating” non-target molecules in a complex sample requires the
convergence of a number of techniques, collectively termed blot transfer.
● Southern (DNA),
● Northern (RNA), and
● Western (protein) blot transfer procedures.

63. Southern hybridization
It is named for the person who
devised the technique [Edward
Southern], and the other names
began as laboratory jargon but
are now accepted terms

64. Southern hybridization

65. Steps of southern hybridization
Step I: Restriction digestion
1. DNA is extracted from the cell.
2. It is partially digested by a restriction endonuclease (RE) that cuts the DNA at a
specific site generating fragments (obtaining complete fragmentation of DNA at
the intended restriction enzyme sites is a critical step in Southern blot analysis).
3. The fragments of DNA obtained by restriction digestion are amplified by PCR.

66. Steps of southern hybridization
Step II: Gel electrophoresis
4. The resulting DNA fragments are separated by electrophoresis. Fragmented
DNA is typically electrophoresed on an agarose gel to separate the fragments
according to their molecular weights. Acrylamide gels can alternatively be used for
good resolution of smaller DNA fragments (<800 bp).
Step III: Denaturation
5. The gel after electrophoresis is then soaked in alkali (NaOH) or acid (HCl) to
denature the double-stranded DNA fragments.

67. Steps of southern hybridization
Step IV: Blotting
6. Without altering their positions, the separated bands of ssDNA are transferred
to a nitrocellulose filter by the process of blotting.
Step V: Baking and blocking
7. After the DNA of interest is bound on the membrane, it is baked on autoclave to
fix in the membrane. The membrane is then treated with casein or bovine serum
albumin (BSA) which saturates all the binding site of membrane

68. Steps of southern hybridization
Step VI: Hybridization with labeled probes
8. The DNA bound to membrane is then treated with labeled probe. The probe
binds with complementary DNA on the membrane since all other nonspecific
binding sites on the membrane are blocked by BSA or casein.
The probe can be labeled with radioactivity, fluorescent dye, or an enzyme that
can generate a chemiluminescent signal when incubated with the appropriate
substrate.

69. Steps of southern hybridization
Step VII: Visualization by Autoradiogram
9. The membrane bound DNA labeled with probe can be visualized under
autoradiogram which give pattern of bands.
10. In the detection step, the bound, labeled probe is detected using the method
required for the particular label used. For example, radiolabeled probes may be
detected using X-ray film or a phosphor imaging instrument, and enzymatically
labeled probes are typically detected by incubating with a chemiluminescent
substrate and exposing the blot to X-ray film.

70. Applications of Southern blot
DNA fingerprinting is an example of southern blotting.
● Used for paternity testing, criminal identification, and victim identification.
● To identify mutation or gene rearrangement in the sequence of DNA.
● Used in diagnosis of diseases caused by genetic defects.
● Used to identify infectious agents.

71. Applications of Southern blot
● Identification of one or more restriction fragments that contain a gene or other
DNA sequence of interest and
● in the detection of RFLPs used in construction of genomic maps.

72. Restriction fragment length polymorphism
● Treatment of genomic DNA from different individuals with a single
restriction enzyme does not always give the same set of fragments
● because some restriction sites are polymorphic,
● being present in some individuals but absent in others,
● usually because a point change in the nucleotide sequence changes
the restriction site into a sequence not recognized by the restriction
enzyme

73. Restriction fragment length polymorphism

74. Northern blotting
The northern blot is a technique used in molecular biology to study gene
expression by detection of RNA (or isolated mRNA) in a sample.
Northern blotting involves the use of electrophoresis to separate RNA samples by
size and detection with a hybridization probe complementary to part of or the
entire target sequence.
They can be DNA, RNA, or oligonucleotides with a minimum of 25 complementary
bases to the target sequence.

75. Northern blotting

76. Western blotting
● The western blot (also called the immunoblot) is a widely used analytical
technique used to detect specific proteins in a sample of tissue homogenate
or extract.
● It uses gel electrophoresis to separate native proteins by 3D structure or
denatured proteins by the length of the polypeptide.
● The proteins are then transferred to a membrane (typically nitrocellulose),
where they are hybridized with antibodies specific to the target protein.

77. Western Blotting

78. Polymerase chain reaction (PCR)
• A molecular technique to copy or amplify small segments of DNA
or RNA.
• An efficient and cost-effective technique that combines the
principles of complementary nucleic acid hybridization with those
of nucleic acid replication that are applied repeatedly through
numerous cycles.
• It results in the exponential production of the specific target
DNA/RNA sequences by a factor of 107 within a relatively short
period.

79. Components of Polymerase Chain
Reactions (PCR)
DNA template (the sample DNA that
contains the target sequence to amplify)
• Deoxyribonucleoside
triphosphates (dNTPs)
• PCR buffer
• Primers (forward and reverse)
• Taq polymerase

80. Steps of procedure
• The PCR amplification occurs by
repeated cycles of three
temperature-dependent steps
called :
• denaturation,
• annealing, and
• elongation

81. Factors affecting PCR
Primer
• A short segment of nucleotides (15-30 bases long) which is
complementary to a section of the DNA or RNA, which is to be
amplified in the PCR.
• Two short DNA sequences designed to bind to the start (forward
primer) and end (reverse primer) of the target sequence are used
in PCR.

82. Forward and reverse primers
The primer that anneals with the
antisense strand or the noncoding
strand or the template strand is
known as forward primer.
Reverse primer is the short DNA sequence that
anneals with the 3’ end of the sense strand or the
coding strand.

83. Factors affecting PCR
Primer (contd.)
• Both reverse and forward primers are important for the production
of millions to billions of copies of particular regions of DNA that are
targeted or interested.
• Hence optimal concentration of primer is needed
• Low concentration of primer results in poor yield while high
concentration may result in non-specific amplification.

84. Factors affecting PCR
ii. Amount of Template DNA
• Optimal amount of template DNA usually
is in nanogram.
• Higher concentrations inhibit or result in
non -specific amplification.

85. Factors affecting PCR
• iii) Taq DNA polymerase:
• A heat-stable DNA polymerase obtained from the thermophilic
bacterium, Thermus. aquaticus is used to synthesize the new strands in
most PCR reactions.
• Since this Taq DNA polymerase works best at around 72°C (161.6°F),
the temperature of the PCR mixture is raised to that temperature for
elongation to proceed efficiently.
• Taq DNA polymerase has both 5’-3’ polymerase and 5’-3’ exonuclease
activities. But it lacks 3’-5’ exonuclease activity (proofreading activity).

86. Detection of PCR products
• Labeled probe that is
specific for the target gene
sequence is used to detect
PCR amplified gene product
(also known as amplicon).
• Based on the nature of the
reporter molecule used,
probe generates radioactive,
colorimetric, fluorometric,
or chemiluminescent
signals.

87. Steps of procedure
• The PCR is carried out in a single test tube containing all
the necessary components.
• The extracted sample (which contains target DNA
template) is added to the tube containing primers, free
nucleotides (dNTPs), and Taq polymerase.
• The PCR mixture is placed in a PCR machine, that
increases and decreases the temperature of the PCR
mixture in automatic, programmed steps and the copies
of the target sequence are generated exponentially.

88. Steps of procedure
1) Denaturation (strand
separation):
• Native DNA exists as a double
helix
• Denaturation separates the two
DNA chains by
• heating the reaction mixture to
90°C to 95°C (194°F to 203°F).

89. Steps of procedure
2) Annealing (primer binding):
• the reaction mixture is cooled to
45°C to 60°C (113°F to 140°F)
• so that the oligonucleotide primers
can bind or anneal to the separated
strands of the target DNA.

90. Steps of procedure
3) Extension (synthesis of new DNA):
During elongation, the DNA polymerase adds nucleotides to the 3
‘ends of the primers to complete a copy of the target DNA template.
These three steps are repeated 20-30 times in an automated
thermocycler that can heat and cool the reaction mixture in tube
within very short time.
This results in exponential accumulation of specific DNA fragments.

91. Steps of procedure
• PCR is a method to amplify any DNA
sequence virtually without limit and allows
the separation of the nucleic acid of interest
from its context.
• The doubling of number of DNA strands
corresponding to target sequences can be
estimated by amplification number associated
with each cycle using the formula.
• Amplification=2n, where n=no. of PCR cycle

92. Steps of
procedure

93. Reverse
transcription PCR
• To carry out polymerase chain
reaction where RNA is the starting
material this method uses reverse
transcriptase, a process called RT–
PCR (reverse transcriptase
polymerase chain reaction).
• The first step in this method is to
convert the RNA molecules into single-
stranded complementary DNA (cDNA).
• After this step, the experiment
proceeds as in the standard technique.

94. RT PCR

96. PCR
• PCR can amplify a desired DNA sequences of any origin hundred
or millions time in a matter of hour, which is very short in
comparison to recombinant DNA technology.
• PCR is especially valuable because the reaction is highly
specific, easily automated and very sensitive.
• It is widely used in the fields like- clinical medicine for medical
diagnosis, diagnosis of genetic diseases, forensic science;
DNA fingerprinting, evolutionary biology.

97. Applications of PCR
1.Forensic science: DNA
fingerprinting, paternity testing
and criminal identification

99. Paternity testing

100. Applications of PCR
2.Diagnosis: Identification and characterization of infectious agents
• Direct detection of microorganisms in patient specimens
• Identification of microorganisms grown in culture
• Detection of antimicrobial resistance
• Investigation of strain relatedness of pathogen of interest

101. Diagnostic significance of PCR
• PCR is becoming the leading method for detection of the continuously
increasing number of human pathogens.
• Examples include:
• Herpes simplex virus (HSV), Human papillomavirus, Human
immunodeficiency virus (HIV), Human T lymphotropic virus type I and
type II,
• Cytomegalovirus (CMV),
• Epstein-Barr virus,
• Human herpesvirus-6 (HHV-6),
• Hepatitis B virus, rubella virus, mycobacteria, Toxoplasmosis gondii,
Trypanosoma cruzi, and malaria.

102. Applications of PCR
3. Detection of mutation
(investigations of
genetic diseases)

103. Applications of PCR
8. Vaccine production by
recombinant DNA
technology

104. Applications of PCR
9. Drug discovery
10. Human genome project

105. Applications of PCR
6. Gene cloning and expression

106. Applications of PCR
7. Gene sequencing

107. Applications of PCR
4. Evolution study: evolutionary
biology
5. Fossil study: paleontology

108. Advantages of PCR over other
diagnostic procedures
1) Sensitivity and specificity- PCR can be applied as a detection method
for virtually any pathogen for which even limited nucleotide sequence
information is known and for which even a very small amount of a
specimen of infected tissue is available.
2) Discriminative- In most cases PCR assays are more discriminative than
conventional serology. For example, it is difficult to distinguish HSV-I
from HSV-II or HIV-1 from HIV-2 by serology, yet such distinctions can
be readily made based on PCR amplification of type-specific genetic
sequences.

109. Advantages of PCR over other diagnostic
procedures
3) PCR yields rapid results, typically in 1 to 2 days in a clinical
setting. The results are specific, faster and accurate
4) It is applicable to a wide variety of clinical, pathologic, or forensic
specimens, as well as to formalin-fixed tissue, inactivated bacterial
cultures, and archaeological specimens.

110. Summary
1) It’s an efficient and cost-effective technique that combines the principles
of complementary nucleic acid hybridization with those of nucleic acid
replication that are applied repeatedly through numerous cycles.
2) The PCR amplification occurs by repeated cycles of three temperature-
dependent steps called denaturation, annealing, and elongation.
3) Labeled probe that is specific for the target gene sequence is used to
detect PCR amplified gene product (also known as amplicon).

111. Summary
4) PCR can be applied as a detection method for virtually any
pathogen for which even limited nucleotide sequence information is
known and for which even a very small amount of a specimen of
infected tissue is available.
5) PCR yields rapid results, typically in 1 to 2 days in a clinical
setting. The results are specific, faster and accurate

112. Thank you