Techniques of molecular biology

1. TECHNIQUES USED IN
MOLECULAR
BIOLOGY
BY
DILIP O. MORANI
ASST. PROF.
SHRI D. D. VISPUTE COLLEGE OF PHARMACY
AND RESEARCH CENTER, PANVEL
1

2. Molecular biology
*Molecular biology, field of science concerned with studying
the chemical structures and processes of biological phenomena
that involve the basic units of life, molecules.
* The field of molecular biology is focused especially on nucleic
acids (e.g., DNA and RNA) and proteins—macromolecules that
are essential to life processes—and how these molecules interact
and behave within cells.
*Molecular biology emerged in the 1930s, having developed out
of the related fields of biochemistry, genetics, and biophysics;
today it remains closely associated with those fields.
2

3. Techniques
• Various techniques have been developed for molecular biology,
though researchers in the field may also employ methods and
techniques native to genetics and other closely associated fields.
• In particular, molecular biology seeks to understand the three-
dimensional structure of biological macromolecules through
techniques such as X-ray diffraction and electron microscopy.
• The discipline particularly seeks to understand the molecular basis of
genetic processes; molecular biologists map the location of genes on
specific chromosomes, associate these genes with particular
characters of an organism, and use genetic engineering (recombinant
DNA technology) to isolate, sequence, and modify specific genes.
• These approaches can also include techniques such as polymerase
chain reaction, western blotting, and microarray analysis.
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4. History
* In its early period during the 1940s, the field of molecular
biology was concerned with elucidating the basic three-
dimensional structure of proteins.
• Growing knowledge of the structure of proteins in the early
1950s enabled the structure of deoxyribonucleic acid (DNA)—
the genetic blueprint found in all living things—to be described
in 1953.
• Further research enabled scientists to gain an increasingly
detailed knowledge not only of DNA and ribonucleic acid (RNA)
but also of the chemical sequences within these substances that
instruct the cells and viruses to make proteins.
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5. Continue…
• Molecular biology remained a pure science with few practical
applications until the 1970s, when certain types
of enzymes were discovered that could cut and recombine
segments of DNA in the chromosomes of certain bacteria.
• The resulting recombinant DNA technology became one of
the most active branches of molecular biology because it
allows the manipulation of the genetic sequences that
determine the basic characters of organisms.
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6. Techniques of molecular biology 6
1. PCR
2. DNA sequencing
3. RFPL
4. cDNA library
5. Blotting techniques
6. Gene therapy

7. 1. PCR
•PCR is a technique that takes
specific sequence of DNA of small
amount and amplifies it to be used
for further testing.
• In vitro technique
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8. Short History of PCR
• 1983: Dr. Kary Mullis developed PCR
• 1985: First publication of PCR by Cetus
Corporation appears in Science.
• 1986: Purified Taq polymerase is first used in
PCR
• 1988: PerkinElmer introduces the
automated thermal cycler.
• 1989: Science declares Taq polymerase
"molecule of the year.
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• 1990: amplification and detection of specific
DNA sequences using a fluorescent DNA-
binding dye, laying the foundation for future
"real-time" or "kinetic" PCR.
• 1991: RT-PCR is developed using a
single thermostable polymerase
facilitating diagnostic tests for RNA
viruses.
• 1993:Dr. Kary Mullis shares Nobel
Prize in Chemistry for conceiving
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10. Continue…
• 1999: Dynal launches DRB-36 HLA-typing
kit for tissue typing.
• 2003: HIV-1 MONITOR Test, version 1.5
Product Family
• AMPLICOR® CT/NG Test for Chlamydia
trachomatis,
• AMPLICOR® CT/NG Test for Neisseria
gonorrhoeae
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11. Purpose
• To amplify a lot of double-stranded DNA
molecules (fragments) with same (identical)
size and sequence by enzymatic method and
cycling condition.
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12. Condition
• 1. Denaturation of ds DNA
template
• 2. Annealing of primers
• 3. Extension of ds DNA
molecules
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13. Denaturation
single
stranded
92 0C
3’
• Temperature: 92-940C
• Double stranded DNA
melts DNA
5’
3’ 5’
+
5’3’
5’ 3’
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14. Annealing
• Temperature: ~50-700C (dependant on the
melting temperature of the expected duplex)
• Primers bind to their complementary
sequences
5’3’
5’ 3’
Forward primer Reverse primer
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15. Extension
• Temperature: ~720C
• Time: 0.5-3min
• DNA polymerase binds to the annealed primers
and extends DNA at the 3’ end of the chain
Taq
5’
3’
Taq5’
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16. Cycling
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17. Products of Extension
3’5’
3’ 5’
3’5’
3’ 5’
Taq
Taq
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18. Overall Principle of PCR
• DNA – 1
copy
• Known sequence Sequence of
interest
Known
sequence
• PC
R
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19. Chemical Components
• Magnesium chloride
• Buffer: pH 8.3-8.8
• dNTPs
• Primers
• DNA Polymerase
• Target DNA
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20. Basic requirements for PCR reaction
• 1) DNA sequence of target region
must be known.
2) Primers - typically 20-30 bases in size.
These can be readily produced by
commercial companies and can also be
prepared using a DNA synthesizer.
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• 3) Thermo-stable DNA polymerase - eg
Taq polymerase which is not
inactivated by heating to 95C.
4) DNA thermal cycler - machine which
can be programmed to carry out heating
and cooling of samples over a number of
cycles.
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22. 22

23. Instrumentation
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24. Three Aspects of PCR
• Specificit
y
• Efficienc
y
• Fidelity
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25. Advantages of PCR
• Small amount of DNA is required per test
• Result obtained more quickly - usually
within 1 day for PCR.
• Usually not necessary to use
radioactive material for PCR.
• PCR is much more precise in
determining the sizes of alleles -
essential for some disorders.
• PCR can be used to detect point
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26. Applications of PCR
• Neisseria gonorrhea
• Chlamydia trachomatis
• HIV-1
• Factor V Leiden
• Forensic testing and many
others
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27. Applications of PCR
Molecular Identification Sequencing Genetic
Engineering
Molecular Archaeology Bioinformatics Site-directed
mutagenesis
Molecular Epidemiology Genomic Cloning Gene Expression
Studies
Molecular Ecology
DNA fingerprinting
Classification of
organisms
Human Genome
Project
Genotyping
Pre-natal diagnosis
Mutation screening
Drug
discovery
Genetic
matching
Detection of pathogens
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28. 2. DNA Sequencing
• Fundamental requirement for modern gene manipulation.
• First DNA sequence was studied of cohesive ends of phage λ
DNA (12 bases long).
• This method was derived from RNA sequencing and not
applicable to large scale DNA sequencing.
• The new method (plus and minus sequencing) was used to
sequence 5386 bp phage.
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• This new method was superseded in 1977 by
chemical degradation method developed by Maxam
and Gilbert and dideoxy chain termination method of
Sanger.
• Methods used for DNA sequencing are classified as
Maxam and Gilbert’s chemical degradation method
and Sanger’s dideoxynucleotide chain termination
method.
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30. Maxam and Gilbert’s chemical
degradation method
• DNA molecule is radio-labelled with 32P at
either its 5’ end by using polynucleotide kinase
or its 3’ end by terminal transferase.
• One end of radio-labelled double stranded DNA
is removed by using endonuclease.
• DNA is then partially modified with chemical
reagents specific for different bases and
cleaved at the modified nucleotides.
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31. Continue…
• This generates a set of molecules differing in
length but with same isotopically labelled
terminus.
• These fragments of different length represent
unique pairs of 5’ and 3’ cleavage products in
the random collection.
• All these fragments are separated by gel
electrophoresis and fragments containing
labelled terminus are observed in the
autoradiogram.
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32. 32

33. Sanger’s dideoxynucleotide
chain termination method.
• This method is more superior than Maxam and
Gilbert’s chemical degradation method
because this method is more efficient and
simple to perform.
• Sanger first developed the method for DNA
sequencing in which utilized DNA polymerase
to extend DNA chain length.
• This method is called plus minus method.
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• Sanger formulated a technique to identify the
DNA sequence by providing a ssDNA solution of
nucleotides of particular type.
• This method is time consuming and not
possible for whole genome sequencing.
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• Sanger developed a more powerful method
utilizing single stranded DNA as the template
for DNA synthesis.
• This method is called dideoxynucleotide chain
termination method.
• Dideoxynucleotides are used as triphosphates
(ddNTP) and can be incorporated in a growing
chain but they terminate synthesis.
• They differ from dNTPs as they lack a –OH
group on the 3’ carbon of the ribose sugar.
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36. Continue…
• Thus, they prevent formation of a phosphodiester bond
with the next nucleotide to be added and prevent further
elongation.
• Different DNA polymerases like E. coli DNA polymerase (I)
genetically modified DNA polymerase from phase T7
(sequence) and Taq DNA polymerase have been used for
sequencing reaction.
• All DNA polymerases require short regions of double
stranded DNA to initiate DNA synthesis on a single stranded
template.
• This is provide by addition of a short ssDNA molecule
known as primer.
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38. Continue…
• For the sequence of DNA molecule, four separate enzyme
reaction are performed in separate reaction tubes.
• Each tube containing template DNA in single stranded form,
DNA polymerase, primer, each of the four deoxynucleotide
triphosphates, dNTPs (dATP, dGTP, dCTP and dTTP) and
modified nucleotide, dideoxynucleotide triphosphate, ddNTPs
(ddATP, ddGTP, ddCTP and ddTTP).
• Concentration of ddNTPs should be maintained to about 1% of
the concentration of dNTPs.
• Ratio of dNTP to ddNTP in the reaction mixture is carefully
adjusted so that a dideoxy moiety is incorporated only
occasionally in place of its deoxy homologues.
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39. Continue…
• All four reactions produce a population of partially
synthesized DNA fragments of different lengths, all sharing
a common 5’ end, all are radioactively labelled and all of
which are terminated by a dideoxynucleotide.
• Synthesized DNA molecules are separated according to size
by electrophoresis on polyacrylamide gels.
• Gel is used for autoradiography so that position of different
bands in each lane can be visualized.
• DNA sequence is obtained by reading the bands on
autoradiogram of four lanes.
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40. 3. Restriction fragment length
polymorphism (RFPL)
• Most restriction enzymes cleave DNA molecules in a site
specific manner.
• Genomic DNA from individual organism is digested with
single or more restriction enzyme and this result in
fragmentation with variation in length.
• These fragments are known as RFLPs.
• DNA exists in fragments of various lengths derived by the
action of restriction enzymes; hence, it is called as
Restriction fragment length polymorphism.
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• Size of restriction fragments can be
determined by agarose gel electrophoresis.
• Single nucleotide polymorphisms (SNPs) can be
detected by sensitive PCR method.
• Variation obtained in one DNA fragment with
that specific enzyme used for digestion is
called one RFLP.
• Detection of RFLPs relies on a specialized
hybridization procedure called southern
blotting.
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42. Continue…
• Extract and purify genomic DNA from different
individuals having some differences and isolate
separately.
• Digest DNA samples by restriction endonuclease
and subject to gel electrophoresis of each
individual on the same gel slab.
• DNA fragments are transferred from gel to
nitrocellulose filters by using southern blotting.
• Filters with small fragments homologous to the
probe can be detected by autoradiography after
hybridization.
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43. Continue…
• Hybridized fragments of genomic DNA is called
RFLP.
• Amplified fragment length polymorphism
(AFLP) is combination of RFLP and random
amplified polymeric DNA (RAPD) which is very
sensitive in detecting polymorphism
throughout the genome.
• RFLP and SNP maps are useful in the human
genome sequencing project.
• It gives information about various single gene
and multigenic diseases.
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44. Applications of RFLP
• It is possible to trace the defective gene by
the analysis of RFLP in DNA.
• RFLP patterns of disease suspected individuals
can be compared with that of normal people.
• RFLPs can also be used to establish linkage
groups by the process of chromosome walking.
• RFLPs are also used to prepare chromosome
maps in rice, tomato, maize, mice, humans
etc.
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46. 4. cDNA library
• mRNAs are highly processed representatives of
genes which express under specific conditions.
• mRNAs cannot be directly cloned because they
are unstable.
• mRNAs are converted to cDNAs and library
made from complimentary or copy DNAs are
called cDNA library.
• cDNA library represents DNA of eukaryotic
organisms.
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47. Continue…
• Genomic DNA of eukaryotes contains introns,
regulatory regions, repetitive sequences, etc.
• Thus, preparation of genomic library is not
meaningful.
• Prokaryotes do not have the ability to remove
the introns.
• Thus, functional mRNA is not correctly formed
in a prokaryotic cell.
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48. Continue…
• In eukaryotic cell, gene is transcribed and RNA
undergoes several changes in nucleus.
• After splicing, functional mRNA is released
into the cytoplasm and introns are removed.
• cDNA library can be prepared by using mRNA.
• mRNA is highly processed, intron free
representatives of DNA having only coding
sequences.
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49. Continue…
• Double stranded cDNA is prepared by following
steps.
i) first strand DNA synthesis on the mRNA
template
ii) removal of RNA template
iii) second strand DNA synthesis using first DNA
strand as a template.
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51. Continue…
• cDNA is a double stranded complement of
mRNA.
• It can be synthesized from mRNA by reverse
transcription.
• mRNA is isolated from animal cell and
purified.
• Oligo dT primer is added to bind to short
segment of poly A tail region.
• Primer provides 3’-hydroxyl group for the
synthesis of a DNA strand.
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52. Continue…
• Enzyme reverse transcriptase and four
deoxynucleotides are added for synthesis of
DNA.
• Newly synthesized DNA strand has a tendency
to fold back to form a hairpin loop.
• Loop of first DNA strand serves as the template
for the synthesis of second DNA strand.
• Second DNA strand is synthesized by addition
of DNA polymerase.
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53. Continue…
• Resulting double stranded DNA fragments are
inserted into a suitable vector and cloned
creating a population of clones called cDNA
library.
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54. Blotting techniques
• Applied in isolation and quantification of
specific nucleic acid sequences and in study of
organization, intracellular localization,
expression and regulation.
• Describe immobilization of sample nucleic
acids on a nylon or nitrocellulose membranes.
• Blotted nucleic acids are then used as targets
in subsequent hybridization experiments.
• DNA, RNA and proteins are easily separated by
blotting method.
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55. Blotting procedures
• These procedures include
i) Southern blotting
ii) Northern blotting
iii) Western blotting
Agarose gel electrophoresis, pulsed field gel
electrophoresis (PFGE), polyacrylamide gel
electrophoresis (PAGE), sodium dodecyl sulphate
polyacrylamide gel electrophoresis (SDS-PAGE)
and two dimensional electrophoresis techniques
are used for separation of DNA, RNA and protein
molecules.
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56. Applications of blotting
• Blotting techniques are widely used as
analytical tools for the specific identification
of desired DNA or RNA fragments.
• Blotting refers to process of immobilization of
sample nucleic acids on solid support.
• Blotted nucleic acids are used as targets in
hybridization experiments for their specific
detection.
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57. Southern blotting
• In 1975, E.M. Southern developed original
method of blotting to identify location of
genes and other DNA sequences on restriction
fragments separated by gel electrophoresis.
• In this technique, sample of DNA containing
fragments of different sizes are subjected to
electrophoresis using either polyacrylamide or
agarose gel.
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• DNA molecule is cut into small fragments by
restriction enzyme and passed through agarose
gel by electrophoresis method.
• It results into separation of DNA molecules
based on their size.
• DNA is then denatured into single strands by
exposing the gel to alkaline solution.
• Gel is added on top of buffer saturated filter
paper.
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59. Continue…
• It is covered with nitrocellulose filter
overlayed with dry filter paper.
• Buffer moves from bottom of filter paper by
capillary action and DNA is trapped in
nitrocellulose membrane.
• This process is known as blotting.
• Nitrocellulose membrane is removed from
blotting stack and DNA is permanently
immobilized on the membrane by baking at
800C or ultraviolet induced cross-linking.
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60. Continue…
• Single-stranded DNA has a high affinity for nitrocellular filter
membrane.
• Then membrane is treated with a solution containing 0.2% each
of Ficoll, polyvinylpyrrolidone and bovine serum albumin.
• This treatment prevents non-specific binding of radioactive
probe.
• Membrane is placed in a solution of labelled RNA, single-
stranded DNA or oligodeoxynucleotide (probe).
• These labelled nucleic acid is used to detect and locate the
complementary sequence, it is called probe.
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61. Continue…
• Probe containing sequence of interest is
hybridized or annealed with immobilized DNA
on the membrane.
• After hybridization reaction, membrane is
washed to remove unbound probes.
• Washed membrane is exposed to X-ray film
that detects the presence of radioactivity in
bound probe.
• Film is developed to reveal bands indicating
positions in the gel of DNA fragments that are
complementary to the radioactive probe.
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62. Applications
• It is very sensitive method and used to map
the reaction sites around a single copy gene
sequence in any genome.
• Used in preparation of RFLP maps, DNA finger
printing, identification of transferred genes
etc.
• Nitrocellulose paper being very fragile is now
replaced by nylon membrane.
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63. 63

64. Northern blotting
• In 1979, Alwine et al devised a technique in
which RNA bands are blot transferred from gel
onto chemically reactive paper.
• Aminebenzyloxymethyl cellulose paper
prepared from Whatman paper 540 after a
series of simple reactions.
• It is diazotized and rendered into reactive
paper.
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65. Continue…
• It becomes available for hybridization with
radio-labelled DNA probes.
• Hybridized bands are found out by
autoradiography.
• Alwine’s method extends that of Southern
method and hence, it has given the jargon
term ‘Northern blotting’.
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66. Continue…
• In 1980, Thomas found that mRNA bands can
also be blotted directly on nitrocellulose paper
under appropriate condition and it can be
hybridized with a labelled DNA or RNA probe.
• Hybrids are treated with S-1 nuclease with
RNAase which digests single stranded DNA/RNA
probe.
• Structure of mRNA is revealed to the extent to
which mRNA protects nucleic acid probe.
• In this technique, preparation of reactive
paper is not required.
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67. Applications
• Mainly useful in studies of gene expression.
• Used to determine whether a particular gene
is transcribed in all tissues of micro-organism
or only certain tissues.
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68. Western blotting
• In 1979, Towbin et al developed western
blotting or protein blotting or electroblotting
technique to find out newly encoded protein
by a transformed cell.
• Polyacrylamide gel electrophoresis is used for
separation and characterization of proteins.
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69. Continue…
• Proteins are extracted from transformed cells
and separated by using sodium dodecyl
sulphate polyacrylamide gel electrophoresis
(SDS-PAGE).
• Sodium dodecyl sulphate acts as a denaturant
for proteins during electrophoresis.
• After electrophoresis, individual proteins are
detected by using specific antibodies and
polypeptides can also be transferred to a
nitrocellulose membrane.
• Transfer of proteins from gels to nitrocellulose
membrane is called western blotting.
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70. Continue…
• It is performed by using electric current,
hence, it is called electroblotting method.
• Electric field is applied to cause migration of
proteins from gel to nitrocellulose filter paper.
• Nitrocellulose membrane is used for probing
with a specific labelled antibody.
• Antibody is labelled with 125I and signal is
detected again with autoradiography.
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71. 71

72. Gene therapy
• Used to treat disease by modifying genetic
information in the cells of the patient.
• Aimed to treat or eliminate the cause of
disease by gene transfer.
• First clinical study was initiated in USA in
1990.
• Desired gene must usually be packed into a
vector system capable of delivering it safely
inside the intended recipient cells.
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73. Vectors used to affect gene
transfer 73

74. Strategies for gene transfer
i) In-vivo approach-involves introduction of
genes directly into target organs of individual
(patient therapy).
ii) ex-vivo approach-cells are isolated for gene
transfer in-vitro followed by transplantation of
genetically modified cells back into the
patients.
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75. Physical delivery systems
• Gene delivery become easy with availability of
many viral, non-viral and physical delivery
systems.
• Retroviruses effectively enter various cell
types and integrate their genome into host cell
genome in stable form.
• Retroviruses are potential vectors for gene
therapy.
• Adenovirus can transfer genes to both
proliferating and quiescent cells and it
efficiently transfers gene by in vivo method.
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76. Approaches for gene transfer
i) Gene modification-replacement therapy,
corrective gene therapy
ii) Gene transfer-physical methods are gene
gun, microinjection, naked DNA,
electroporation etc. chemical methods as
oligonucleotides, cationic liposomes etc. and
biological methods as mammalian artificial
chromosomes, viral vectors etc.
iii) Gene transfer in specific cell lines-somatic
gene therapy, germline gene therapy.
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77. Genetic diseases
• More than 4000 genetic diseases have been
characterized and studied.
• These diseases are caused by lack of
production of a single gene product or due to
production of a mutated gene product.
• Slow progress to treat genetic diseases is likely
due to number of factors.
• Identification of actual gene responsible for
disease, proper gene delivering vectors,
complexity of disease, limited patient
population etc. are major problems for
development of gene therapy.
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79. Clinical trials
• Most of clinical trials involving gene therapy
have been directed to tumor cells.
• Introduction of TNF gene into tumor in-
filtrating lymphocytes, insertion of a copy of a
tumor suppresser gene into cancer cells and
killing of tumor cells by transfection with
thymidine kinase gene of herpes simplex virus
are major techniques used for gene therapy in
cancer.
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80. Reference
• Pharmaceutical biotechnology- Fundamentals
and Applications by Prof. Chandrakant Kokare,
Sixth edition-August 2018, Nirali Prakashan.
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