Textbook : Petter_Laake,_Haakon_Breien_Benestad,_Bjorn_Reino

1. RESEARCH
METHODOLOGY
CHAPTER 6: METHODS OF
MOLECULAR BIOLOGY

2. 6.1 INTRODUCTION
Molecular biology can be defined as the scientific discipline covering
the level between chemistry and biochemistry on the one side and
cell biology, histology, anatomy and physiology on the other,
intertwined with and overlapping the levels on each side.

3. RECOMBINANT TECHNOLOGY
The foundation was laid in the 1960s, with the discoveries of:
• Restriction endonuclease (RE) enzymes - to cut DNA at specific
sites into shorter fragments amenable to analysis
• The techniques of DNA denaturation/renaturation and
hybridization - by which specific pieces of DNA could be identified
and pulled out from a large pool of cut fragments
• The enzyme DNA ligase - allowed joining of fragments together
into new combinations.

4. 1970’S
 DNA cloning was made feasible in the early 1970s.
 Marked the birth of recombinant DNA technology.

5. 6.2 RESTRICTION ENDONUCLEASES
 Endonucleases are enzymes that cleave the phosphodiester bonds
of the DNA or RNA backbone
 Some restriction endonucleases cut the two strands immediately
opposite one another, generating blunt ends,
 staggered cuts, generating single-stranded overhangs referred to
as sticky ends

6. VECTORS*
Plasmid vectors
Vector is a
1. Carrier (medicine)
2. DNA molecules with the capacity to replicate
when introduced into host cells and onto
which we can load pieces of DNA to be
amplified (Molecular Biology)
 Polylinker or multiple cloning site are several
different recognition sites put one after the
other within a small region.
 A shuttle vector is a vector (usually a plasmid)
constructed so that it can propagate in two
different host species
Three elements are shared by all plasmid vectors
1. An origin of replication, i.e. a sequence
necessary for replication in the bacterial host;
2. A cloning site containing recognition
sequences for one or more restriction enzymes,
wherein the desired DNA fragment can be
inserted.
3. A selection marker - a gene conferring a
phenotype that can be selected for or against,
usually a sequence conferring resistance to
antibiotics

7. Viral Vectors
 Viruses are protein-coated infectious DNA or RNA
particles with naturally evolved mechanisms for
unassisted entry into host cells.
 Bacteriophages are viruses that infect and
replicate in bacteria
 Specialized to host cells they can infect ( tropism)
 Viral vectors : Advantage - to transfer genes into
intact multicellular organisms. E.g. rats
Disadvantage - random sites of genomic
integration, they generate the hazard of
unwanted spread.
Transferring DNA into host cells –
Conjugation - process for genetic exchange between
bacteria
Transduction – Transfer genetic information between
cells
Transformation is the genetic alteration of bacterial
cells as a result of uptake of foreign DNA
or RNA.
Transfection - Non-viral transfer of DNA into
eukaryotic cells
Transformation - refers to the changes occurring when
eukaryotic cells become malignant
Bacteria can spontaneously take up non-viral nucleic
acids from the surroundings. Methods used to
regulate uptake are; Electroporation, or by adding Ca2
or other divalent cations to the medium

8.  To insert non-viral DNA into eukaryotic cells the
used techniques are:
1. Electroporation,
2. Injection directly into the cell (microinjection)
3. Complexing the DNA with molecules that
facilitate the DNA transfer
In bacterial transformation, the new DNA is
normally not integrated into the bacterial
chromosome, but remains as an extrachromosomal
plasmid
 In eukaryotic cells, transfection may be transient
or stable.
 Transient transfection, many copies of the
DNA are transferred into each cell, where they
remain extrachromosomally and are expressed
only for a short period, usually a few days
 Stable transfection, the foreign DNA, which is
either integrated into chromosomal DNA or
stays extrachromosomally, is maintained by
selection for resistance to cytotoxic drugs

9. Escherichia coli
 The laboratory workhorse
 Its full genome sequence (approx. 4.6 million bp)
was reported in 1997
 Divides rapidly
 Exclusively the bacterial species used in DNA
technology
 strains divide every 20–30 minutes then lag
phase. ( Continuous division is log phase)

10. 6.3 THE TECHNIQUES OF DNA DENATURATION/RENATURATION AND
HYBRIDIZATION
DNA AND RNA: ISOLATION, IDENTIFICATION, SYNTHESIS AND ANALYSIS
Isolation of DNA and RNA
 DNA and RNA are negatively charged
 Not soluble in organic solvents (phenol and
chloroform)
 Soluble in water
The phenol/chloroform isolation method
disadvantages:
 It is time consuming
 involves flammable and toxic liquids
 Replaced by chromatographic methods
Chromatographic method steps ( DNA/RNA
Purification)
1. Anion-exchange resins consist of positively
charged solid particles, to which the highly
negatively charged DNA and RNA molecules
binds strongly.
2. Sugars and proteins, which bind with moderate
affinity, can be washed off the resin
3. Highly purified nucleic acids are then eluted
using buffers with the appropriate pH and salt
concentration

11. Purification Method ( Density Difference)
 DNA/RNA purification is based on
differences in densities between RNA,
supercoiled DNA, linear DNA, proteins and
lipid (with densities decreasing in the order
listed).
 Use of ultracentrifugation in caesium
chloride density gradients
 When working with nucleic acids it is important
to avoid degradation due to contamination with
DNases and RNases

12. IDENTIFICATION – (Visualization: Northern and Southern Blots)
DNA and RNA molecules can be separated for
analytical and preparative purposes by:
 Electrophoresis in agarose or
 polyacrylamide gels (used in sequencing,
mutation detection and separation of short
fragments)
 DNA and RNA are visualized in the gel using
fluorescent dyes such as ethidium bromide.
 Distinct fragments can be identified based on
their nucleic acid sequence with a DNA probe
Complementary strands – two strands with same
base pairing
 Denatured or separate into single strand by
heating, immersion in denaturing buffers, or
both
 Renature or rejoin by cooling or changing
buffers.
How its visualized?
a probe with sequence matching one of the
fragments can be added to single-stranded target
DNA which forms a hybrid together with the target
DNA fragment that has a complementary sequence
(for visualization it is labelled with radioactive
nucleotides (32P, 33P or 35S)…

13. VISUALIZATION CONT.
…that can be detected by autoradiography or
haptens that can be detected by staining with
antibodies.
Southern Blot
 first described by E. M. Southern
 To preserve the electrophoretic separation of the
molecules during denaturation and hybridization,
the DNA (or RNA) must be immobilized by blotting
onto a membrane with high binding capacity for
nucleic acids.
 The method for detection of specific,
electrophoretically separated DNA fragments is
consequently called Southern blot
 RNA is Northern Blot and Proteins Western Blot
 DNA/RNA may also be applied to membrane for
detection by hybridization with probes without
prior electrophoretic separation.
 Dot blot pipetted directly onto the membrane

14. SYNTHESIS
 plaque lift is the transfer from virus colonies to
membrane
 Colony lift is the transfer from bacterial
colonies.

15. Synthesis: The polymerase chain
reaction
 PCR is used to amplify only specific segments
of DNA.
 DNA polymerases enzyme is used to generate
copies of DNA in cyclically which doubles
output.
 DNA polymerases require a primer at the 5
side
 Amplified regions can be controlled by adding
synthetic DNA primers.
 PCR takes place entirely outside living cells
 What is needed for PCR ?
1. Template DNA,
2. Primers,
3. The four deoxynucleotides (in triphosphate
form dTTP
, dCTP
, dATP and dGTP),
4. DNA polymerase
5. Suitable buffer

16. The reaction is repeated in a cycle consisting of three steps:
1. DNA denaturation, where the reaction mix is heated to 94°C,
causing separation of the two DNA template strands
2. Annealing, where the temperature is lowered so that the
primers can hybridize with the template DNA. Because the
primers are present in huge surplus, they will hybridize with the
template before renaturation of the template DNA
3. Elongation, which is performed at the optimal working
temperature of the DNA polymerase, usually around 72°C.
 Taq is efficient and robust,
but with a high error rate
as it lacks 3 to 5
exonuclease activity

17. Purpose of PCR
 In diagnostics, to identify the presence of a
particular sequence (e.g. belonging to viruses or
bacterial strains) in a sample.
 In cloning of cDNA (see below) or of fragments of
genomic DNA. The pieces of DNA are first
amplified by PCR, and can then be ligated into a
plasmid vector. Pieces of DNA encoding parts of
different proteins may be ligated together into the
plasmid, for generation of chimaeric proteins. The
purpose may also be construction of plasmids
with novel properties.
 For quantification of mRNA: see reverse
transcriptase (RT)–PCR, below.

18. ANALYSIS: DNA sequencing (i)
 The key element developed by Sanger and co-
workers, is the use of dideoxynucleotides that
when incorporated into the newly synthesized
DNA strand terminate further elongation
Reverse transcription and cDNA libraries
(ii)
 Reverse transcriptases catalyse the synthesis of
DNA using RNA as a template
 . A collection of cDNA synthesized from the
whole set of isolated mRNAs is referred to as a
cDNA library
 . cDNA libraries are therefore cell, tissue and
organ specific.

19. Artificial chromosomes and genomic
libraries
 Libraries can also be made from genomic DNA
 Yeast artificial chromosomes (YACs) permit
cloning of DNA fragments exceeding a million
base pairs. YACs are technically difficult to work
with, and a serious disadvantage is their
tendency to recombine
 Phage- and bacteria-derived artificial
chromosomes (PACs and BACs
Differences between cDNA libraries and
genomic libraries
 cDNA libraries - are made from mRNA and will
therefore vary according to cell or tissue source.
 Genomic libraries - are made from genomic
DNA.

20. 6.4 PRACTICAL APPLICATIONS OF DNA/RNA TECHNOLOGY
1. Whole genome sequencing
2. Identification of novel genes
3. Analysis of gene expression
4. Semiquantitative and quantitative reverse
transcriptase-polymerase chain reaction
5. Microarrays
Whole Genome sequencing
 The assembly problems led to two different
main strategies for genome sequencing
1. Human Genome Project is the hierarchical
shotgun sequencing two-step method (multi
step)
2. whole genome shotgun sequencing (single
step)

21. Identification of novel genes
 Novel genes are primarily identified by computer
analysis (in silico cloning)
 Two main approaches are used:
1. The first predicts the existence of genes ab
initio. (without previous knowledge other than
general rules for how genes are organized, and
applies primarily to protein encoding genes)
2. The second is based on sequence similarities
(homology) with genes already known.
The input sequence can be used to search for the
corresponding (orthologous) gene in a different
species or for a related but different (homologous)
gene in the same species. Duplicated genes in the
same species are called paralogs
Search tools used to identify novel genes expression
are:
I. Basic Local Alignment Search Tool (BLAST)
program
II. Homology screening is also performed in vitro,
based on hybridization to DNA probes and with
cDNA libraries usually ligated into bacteriophage
λ-derived vectors
III. expression cloning
IV. subtraction cloning
V. positional cloning

22. Analysis of gene expression
 What make cells different is the different
selections of genes they transcribe and express
 Investigations of gene expression are based mainly
on measurements of transcriptional activity
 Northern blot , is the traditional method for
quantitative measurements of RNA
 Disadvantages
1. time consuming,
2. requires relatively large amounts of RNA and can
give spurious results owing to cross-hybridization
Semiquantitative and quantitative
reverse transcriptase-polymerase chain
reaction
 Disadvantages
1. The near-exponential characteristics of the
amplification make it vulnerable
2. small differences in reaction conditions between tubes
can have large effects
3. The number of cycles must be limited to avoid
exhausting the reagents but still sufficient for reliable
detection of PCR product
4. Because only the endpoint of the reaction is measured,
the quantitative accuracy of the method is limited
With the use of internal controls to normalize for sample to
sample variations in total RNA amounts and reaction
efficiency, it is possible to reach reasonable,
semiquantitative estimates of specific RNA in the samples

23. Semiquantitative and quantitative
reverse transcriptase-polymerase chain
reaction
 Advantages
1. It does not require sophisticated apparatus,
2. and is rapid,
3. sensitive and,
4. Highly specific (depending on the appropriate
choice of primers)
5. Being replaced by real-time or quantitative PCR
(qPCR) (fluorescent intensity is measured)
Microarrays
This systematic deposition of probes is called a
microarray
Disadvantage
1. The electrophoretic separation of mRNA in the
northern blot technique is time consuming and
limits the number of samples that can be
processed simultaneously
Advantages
1. It yields information about the lengths of the
mRNA to which the probe hybridizes
2. Ability to analyse expression of thousands of
genes in a single mRNA sample.

24. Two-channel microarrays cDNA made
from two different samples
The system allows detection of
upregulated or downregulated genes
in the test sample versus the control,
but does not give information about
absolute expression levels
Difference between the northern
blot (or its simplified dot-blot
equivalent) and the microarrays
Northern Blot - monitors the
expression profile of a single gene in
different tissues
Microarrays - the expression profile of
thousands of genes in a single tissue
(or compares two tissues)

25. FUNCTIONAL ANALYSIS OF THE ENCODED PRODUCTS
 Vectors for functional studies: expression
vectors
The vector must contain suitable promoters (expression
vectors), ensuring efficient transcription in the particular
cell type under study
 Modifying the inserted genes can be
informative
 Gene knockdown by targeting specific mRNAs -
elucidating the function of a protein when it is no
longer present, or its expression is significantly
reduced.
• knockdown experiment, and is performed by
specifically targeting the corresponding mRNAs for
degradation, stopping them from being translated.
• The second method is referred to as siRNA (small
interference RNAs), exploiting an evolutionary
 Transgenic and knockout animals
Techniques for genetic manipulation of experimental
animals
1. Transgenic - a gene is transferred into the
genome by the use of retroviral vectors or by
microinjection at the zygote (fertilized egg) stage.
the gene is inserted at more or less random sites in
the chromosomes. The transgene is therefore
heritable
2. knockout animals - are generated by
experimental inactivation of one or more
chromosomal genes.

26. IDENTIFICATION OF GENES ASSOCIATED WITH DISEASE: SINGLE
NUCLEOTIDE POLYMORPHISM TYPING
 In medicine, identifying disease genes is of
utmost value for diagnostic, therapeutic and
preventive purposes.
 SNP - single nucleotide polymorphisms. Are
alterations of single nucleotides in the genome,
constitute the main source of genetic variation
between two individuals of the same species
 International HapMap Project – used to
identify SNPs inherited together
 Simple tandem repeats (STR) - highly useful as
DNA markers in family linkage studies as well as
in forensic medicine for determining paternity,
clarifying alleged kinships in cases of illegal
immigration and for DNA fingerprinting in
criminal cases.

27. 6.5 PROTEIN ANALYSES
 The main challenges are:
• the wide range of physical
properties, with water solubility as a
major problem
• the wide range in protein
concentrations, which for cellular
proteins may vary with a factor of 106
(e.g. actin versus transcription factors)
and for serum proteins with a factor of
109 (albumin versus cytokines)
• the problem of making a comprehensive set
of reporter ligands or probes for
quantification by protein arrays or mapping of
cell and tissue localization
• determinations of three-dimensional (3D)
structures, which currently cannot be
predicted, but must be determined by
resource-demanding techniques, mainly X-ray
crystallography and nuclear magnetic
resonance
• the complexity of interactive networks
between proteins.

28. Protein purification
Methods for protein purification exploit
• Differences in surface properties, such as
distribution and accessibility of charged, polar
and hydrophobic groups, which results in
differences in solubility in solvents where
variables are salt concentration/ionic strength,
pH and content of detergents (precipitation)
• Differences in size and shape (gel filtration, SDS
gel electrophoresis)
• Net charge (ion-exchange chromatography,
isoelectric focusing)
• Biological properties, mainly affinity for other
molecules (affinity chromatography with natural
ligand or specific antibody bound to solid
phase).

29. Protein expression and recombinant
proteins
 Protein expression refers to the directed
synthesis of a large amount of a specific protein.
 The main advantage of E. coli is its simplicity in
use: it is easily transformed, divides rapidly,
thrives in cheap culture media and can produce
large amounts of the desired protein.
 The disadvantage is that the protein may not
be correctly folded or modified.
Protein analyses by electrophoresis,
western blotting and
immunoprecipitation
 Proteins can be separated by electrophoresis.
 Detection of protein can be done unspecifically
in the gel by staining methods
 Visualization of a specific protein is commonly
done by transferring the proteins in the gel to a
polyvinylidene fluoride (PVDF) membrane, a
process known as western blotting
 One popular use of western blot is the
identification of protein interactions

30. Two-dimensional electrophoresis
 Proteins can be separated based on their net
charge.
 Every protein has an isoelectric point (pI), that is,
a pH at which its net charge is zero
Protein–protein interactions
 One way of identifying such interacting proteins
is immunoprecipitation followed by western
blot or protein sequencing
 Proteomics - collection of proteins that are
expressed at a given time in an organism, a
tissue or a cell

31. Expression proteomics and protein
expression arrays
Methods used should exhibit
• high resolving power, including the ability to
distinguish between modified variants of the same
protein
• high sensitivity
• suitability for high-throughput analysis
• the ability correctly to reflect relative protein
concentrations.
Mass spectrometry
 Mass spectrometry, - separates ionized
molecules according to mass to charge (m/z)
ratios
 The protein spots of interest are cut out of the
2D gel and digested with an endopeptidase,
usually trypsin, which cleaves the C-terminal to
the basic amino acids lysine and arginine,
generating peptides of suitable lengths for
further analysis

32. In situ mapping of proteins
 The main reporter ligands for in situ mapping of
protein are mAbs
 Quantum dots are tiny semiconductors that can
replace organic fluorescent dyes as reporter
tags.

33. 6.6 BIOINFORMATICS
 Bioinformatics is ‘The science of managing and analysing
biological data using advanced computing techniques’,
and emphasizes its pivotal role in analysing genomic
research data

34. APPLICATIONS OF BIOINFORMATICS
 virtual evolution,
 metabolic networks,
 molecular 3D predictions
 morphometrics

35.  . UniProt (the Universal Protein Resource) is a databank dedicated
to information and analysis of proteins.