3 Nucleic Acid Hybridization and Amplification PDF
3 Nucleic Acid Hybridization and Amplification PDF
3 Nucleic Acid Hybridization and Amplification PDF
MODULE 3- LECTURE 1
METHODS OF NUCLEIC ACID DETECTION
3-1.1 Introduction:
Nucleic acid molecules like deoxyribonucleic acids (DNA), ribonucleic acids (RNA) are
basic, essential and primary molecules for all molecular biology related research. Before
discussing the common methods to detect nucleic acids let us we learn about the isolation
of pure form of nucleic acid.
3-1.3.1 UV spectroscopy
Majority of bio-molecules intrinsically absorb light in the ultraviolet and not in the
visible range. This property of UV absorbance can be used to quickly estimate the
concentration and purity of DNA and RNA (also proteins) in a analytical sample. The
amount of DNA in a sample can be estimated by looking at its absorbance at a
wavelength of 260nm or 280nm (in the UV region). Purines and pyrimidines have
absorbance maxima slightly below and above 260 respectively. Thus the absorbance
maxima of different fragments of DNA vary somewhat depending on their subunit
composition. Contaminants like proteins exhibit two absorbance peaks, one between 215-
230 nm (due to peptide bonds absorption) and at about 280 nm (absorption by aromatic
amino acids-tyrosine, tryptophan and phenylalanine). Remember that although proteins
have little absorbance at 260 nm, both proteins and nucleic acids absorb light at 280 nm.
That is the reason why, if nucleic acids and proteins are mixed in the same sample, their
spectra interfere (overlap) with one another.
Table: 3-1.1 The relationship between concentration of DNA, RNA, Protein and
absorptivity are as below:
1
"Antibodies: A Laboratory Manual", by E. Harlow, and D. Lane, p. 673. Academic
Press, New York., 1988.
The purity of a solution of nucleic acid is determined by measuring the absorbance of the
solution at two wavelengths, usually 260 nm and 280 nm, and calculating the ratio of
A260/A280. Value of this ratio is 2.0, 1.8and 0.6 for pure RNA, DNA and protein
respectively. A ratio of less than 1.8 signifies that the sample is contaminated with
protein or phenol and the preparation is not proper.
Incident Light
Unabsorbed
Li ht
Detector
UV (260nm)
UV (260nm) Nucleic Acid
Sample
Fig. 3-1.3.1.1 -Nucleic acid melting curve showing hyperchromicity as a function of temperature
Fig 3-1.3.2 Ethidium bromide Fig. 3-1.3.2.1 Agarose gel stained with EtBr
Ethidium bromide (EtBr) is a potent mutagen and carcinogen. Dyes to stain nucleic
acids such as SYBR green, SYBR Safe etc are safer to use instead of EtBr.
Polyacrylamide gel can be used for separation of Nucleic acids and post staining of the
gel with EtBr is done for detection. The sensitivity of EtBr staining is of nano-molar
level.
Silver staining based on reduction of silver nitrate is more sensitive than ethidium
bromide for double stranded DNA, as well as detection of single stranded DNA or RNA
with a good sensitivity (in picogram level). It is based on the reduction of silver cations to
insoluble silver metal by nucleic acids. This chemical reaction is insensitive to the
macrostructure of the DNA molecule. Reduced silver molecules deposit in the gel around
the DNA bands, creating a dark black band like image (i.e. “latent image”). Then the
latent image can be developed to visualize by soaking the gel in a solution of silver
cations (Silver nitrate) and a reducing agent (eg. formaldehyde). The silver granules in
the latent image catalyze the further reduction and deposition of silver from the solution.
Bands manifest as dark brown or black regions which appear before significant
background develops. Development is stopped by altering the pH of the gel to a point
where silver reduction is no longer favored.
3-1.3.4 Nanodrop:
Detection assays are persistently being developed that use progressively smaller
amounts of nucleic acid, often precluding the use of conventional cuvette-based
instruments for nucleic acid quantitation for those that can perform micro-volume
quantitation. The patented NanoDrop microvolume sample retention system functions
by combining fiber optic technology and natural surface tension properties to capture
and retain small amounts of sample . This is a novel technology which allows us to
measure nano-liter volumes (pico concentration) of the nucleic acid (DNA or RNA)
sample. It is a type of spectrophotometer with a smaller sample size (as much less as 1-2
microlitre) requirement and higher sensitivity (even upto pico molar level). This is also a
time saving technology widely used in basic molecular biology research.
(Source: http://www.biotech.wisc.edu/facilities/gec/equipment/nanodropnd1000)
Fluorometric method applies fluorescence dyes to detect the presence and concentration
of a class of nucleic acid (DNA or RNA). This method is more sensitive and less prone to
contaminants than UV spectroscopy.
An assay using Hoechst 33258 dye is specific for DNA because it is less sensitive to
detect RNA. This assay is commonly used for rapid measurement of low quantities of
DNA, with a detection limit of ~1 ng DNA. It is useful for the measurement of both small
and large amounts of DNA (verifying DNA concentrations prior to performing
electrophoretic separations and Southern blots) because this assay accurately quantifies a
broad range of DNA concentrations from10 ng/ml to15 μg/ml. The Hoechst 33258 assay
can also be employed for measuring products of the polymerase chain reaction (PCR)
synthesis.
Hoechst 33258 is non-intercalating reagent and binds to the minor groove of the DNA
with a preference for AT sequences (Portugal and Waring, 1988). The binding to the
minor groove has is dependent upon a combination of structural preferences (eg., the
minor groove with a series of contiguous AT base pairs is more narrow).(Neidle (2001)
,Like other minor groove binding ligands, Hoechst 33258 is positively charged and thus
form electrostatic interaction with the negative potential of stretch of AT base pairs.
Upon binding to the minor groove of the double helix DNA, the fluorescence
characteristics of Hoechst 33258 change dramatically, showing a large increase in
emission at ~458 nm.
According to Daxhelet et al, the fluorochrome 4’,6-diamidino-2-phenylindole (DAPI) has
similar characteristics to H33258 and binds to the minor groove as well. DAPI is also
appropriate for DNA or RNA quantitation, although it is not as commonly used as
Hoechst 33258. DAPI is excited with a peak at 344 nm. Emission is detected at around
466 nm for DNA, similar to Hoechst 33258 but for RNA the peak shifts to ~500 nm.
Hybridization-Based Techniques for nucleic acid detection has higher resolution (down
to the actual nucleotide sequence) and utilizes "probe sequence" for DNA or RNA, and
when it finds its intended target, binds to it by hybridization process. Since the "probe" is
attached to a label such as a fluorescent chemical isotope, the bound sequence can thus be
visualized. This is the principle of Southern Blotting method, as well as its variants, and
requires the target nucleic acid sequence to undergo separation by agarose-
electrophoresis, transferred to an appropriate membrane (typically nitrocellulose), and
then treated with a solution containing the labeled probe (fluorescent or colorimetric).
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. Since the probe specifically binds to its target, the membrane can be
documented and analyzed so that the bound target sequences can be identified and
studied. The detailed methodology is described in later chapter (Module-4Lecture 3).
Bibliography:
Bauman, J. G., Wiegant, J., Borst, P. and van Duijn, P. (1980). A new method for
fluorescence microscopical localization of specific DNA sequences by in situ
hybridization of fluorochrome labelled RNA. Exp. Cell Res. 128, 485-490.
Daxhelet, G.A., Coene, M.M., Hoet, P.P., and Cocito, C.G. 1989. Spectrofluorometry of
dyes with DNAs of different base composition and conformation. Anal. Biochem.
179:401-403.
Fluorometric Quantitation of DNA Using Hoechst 33258 Cold Spring HarbProtoc; 2006;
doi:10.1101/pdb.prot4458.
Kislauskis, E. H., Li, Z., Singer, R. H. and Taneja, K. L. (1993). Isoform specific
3’untranslated sequences sort alpha-cardiac and beta-cytoplasmic actin messenger RNAs
to different cytoplasmic compartments. J. Cell Biol. 123, 165-172.
Neidle, S. DNA minor-groove recognition by small molecules. 2001. Nat. Prod. Rep.
18:291-309.
Portugal, J. and Waring, M.J. (1988). Assignment of DNA binding sites for 4,6-
diamidine-2-phenylindole and bisbenzimide (Hoechst 33258): A comparative
footprinting study. Biochem. Biophys. Acta 949:158-168.
MODULE 3- LECTURE 2
POLYMERASE CHAIN REACTION (PCR) AND ITS
APPLICATIONS
3-2.1 Introduction:
Polymerase chain reaction (PCR) is a widely employed technique in molecular
biology to amplify single or a few copies of DNA, generating millions of copies of a
particular DNA sequence. The polymerase chain reaction results in the selective
amplification of a target region of a DNA or RNA molecule. PCR has been extensively
exploited in cloning, target detection, sequencing etc. The method consists of thermal
cycles of repeated heating followed by cooling of the reaction mixture to achieve melting
and primer hybridization to enable enzymatic replication of the DNA.
3-2.2 History:
By 1971, a “repair synthesis" process was reported which was an artificial system
containing primers and templates that can allow DNA polymerase to copy target gene.
The DNA polymerases initially employed for in vitro experiments were unable to
withstand these high temperatures. In 1976, Chien et al discovered a novel DNA
polymerase from the extreme thermophile Thermus aquaticus which naturally dwell in
hot water spring (122 to 176 °F). The enzyme was named as Taq DNA polymerase which
is stable upto 95°C. In 1985, Kary Mullis invented a process Polymerase Chain Reaction
(PCR) using the thermo-stable Taq polymerase for which he was awarded Nobel Prize in
1993.
2. Primers that are complementary to the 5' ends of each of the sense (Forward primer)
and anti-sense strand of the DNA target (Reverse primer).
3. Taq polymerase or other thermostable, high fidelity DNA polymerase (Pfu polymerase
isolated from Pyrococcus furiosus).
5. Buffer solutions to provide a suitable chemical condition for optimum activity and
stability of the DNA polymerases.
6. Divalent cations (eg. magnesium or manganese ions). They act as a co-factor for Taq
polymerase which increases its polymerase activity. Generally Mg2+ is used, but Mn2+
can be applied to achieve PCR-mediated DNA mutagenesis. This is because higher Mn2+
concentration leads to higher error rate during DNA synthesis.
3-2.3.2 Procedure:
Typically, PCR is designed of 20-40 repeated thermal cycles, with each cycle
consisting of 3 discrete temperature steps: denaturation, annealing and extension. The
thermal cycles are often proceeded by a temperature at a high range (>90°C), and
followed by final product extension or brief storage at 4 degree celsius. In PCR cycles,
the temperatures and the duration of each cycle is determined based on various
parameters like the type of DNA polymerase used, the melting temperature (Tm) of the
primers, concentration of divalent ions and dNTPs in the reaction etc. The various steps
involved are:-
a) Initial Denaturation
b) Denaturation
c) Annealing
d) Extension
e) Final extension
T
Repeat Step 1-3
e
Step 1 Step 2 Step 3 Begin Step 1 for 25-30 cycles
m
p
e 5
10 –fold
0
r ( C) 94
amplification of
a Heat Synthesis of
72 Primer target DNA
t Denaturation complementary
annealing fragment
chain
u 55 After 30 cycles
r 0
hold at 4 C
e 22
1 2 3 4 5
Time (min)
Initial
Denaturation
INITIALIZATION Step
3-2.3.2.2 Denaturation:
Denaturation requires heating the reaction mixture to 94–98 °C for 20–30
seconds. It results in melting of the DNA template by disrupting the hydrogen bonds
between complementary bases, yielding single-stranded DNA molecules.
3-2.3.2.3 Annealing:
Following the separation of the two strands of DNA during denaturation, the
temperature of the reaction mix is lowered to 50–65 °C for 20–50 seconds to allow
annealing of the primers to the single-stranded DNA templates. Typically the annealing
temperature should be about 3-5 °C below the Tm of the primers. Stable complimentary
binding are only formed between the primer sequence and the template when there is a
high sequence complimentarity between them. The polymerase enzymes initiate the
replication from 3’ end of the primer towards the 5’end of it.
3-2.3.2.4 Extension/Elongation:
Extension/elongation step includes addition of dNTPs to the 3’ end of primer with
the help of DNA polymerase enzyme. The type of DNA polymerase applied in the
reaction determines the optimum extension temperature at this step. DNA polymerase
synthesizes a new DNA strand complementary to its template strand by addition of
dNTPs, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at
the end of the nascent (extending) DNA strand. Conventionally, at its optimum
temperature, DNA polymerase can add up to a thousand bases per minute. The amount of
DNA target is exponentially amplified under the optimum condition of elongation step.
The drawback of Taq polymerase is its relatively low replication fidelity. It lacks a 3' to 5'
exonuclease proofreading activity, and has an error rate measured at about 1 in 9,000
nucleotides.
Final hold step at 4 °C may be done for short-term storage of the reaction mixture.
The specificity of the PCR depends crucially upon the primers. The following factors are
important in choosing effective primers.
b) Ideally GC content of the primers is 50% .For primers with a low GC content, it is
desirable to choose a long primer so as to avoid a low melting temperature.
Tm = 4°C x (number of G’s and C’s in the primer) + 2°C x (number of A’s and T’s
in the primer)
Two primers must have a similar Tm value. In case of several primer candidates, we
have to choose primers which have the higher Tm value among them.
d) Sequences with long runs (i.e. more than three or four) of a single nucleotide should be
avoided.
e) Primers with significant secondary structures (self –hair pin, loop formation) are
undesirable.
3-2.5 Applications
PCR-based diagnostics tests are available for detecting and/or quantifying a number of
pathogens, including:
6.Cytomegalovirus, might cause life threatening disease in transplant patients and other
immunocompromised people, including HIV-1/AIDS patients
7.Mycobacterium tuberculosis, which in its active state causes tuberculosis and can lead
to tissue damage of infected organs.
Genetic counselling is done for the parents to check the account of genetic disease
beforehand to make a decision on having children. This is of course governed by national
laws and guidelines. Detection of genetic disease before implantation of an embryo in
IVF (In vitro fertilisation) also known as pre-implantation diagnosis can also be done
exploiting PCR based method. Further to diagnose inherited or a spontaneous disease,
either symptomatic or asymptomatic (because of family history like Duchene muscular
dystrophy) PCR based method is very useful.
3-2.5.6 Others
PCR has numerous applications in various fields. The Human Genome Project
(HGP) for determining the sequence of the 3 billion base pairs in the human genome,
relied heavily on PCR.The genes associated with a variety of diseases have been
identified using PCR. For example, Duchenne muscular dystrophy,which is caused by the
mutation of a gene, identified by a PCR technique called Multiplex PCR. PCR can help
to study for DNA from various organisms such as viruses or bacteria. PCR has been used
to identify and to explore relationships among species in the field of evolutionary
biology. In anthropology, it is also used to understand the ancient human migration
patterns. In archaeology, it has been used to spot the ancient human race. PCR commonly
used by Paleontologists to amplify DNA from extinct species or cryopreserved fossils of
millions years and thus can be further studied to elucidate on.
Bibliography:
Module 3: Lecture 3
VARIATIONS IN PCR AND THEIR APPLICATIONS
3-3.1 Introduction:
In the last lecture we discussed that in 1985 Kary Mullis developed Polymerase
chain reaction (PCR) which has become an everyday laboratory procedure to obtain
millions of copies of a specific piece of DNA of interest. The reaction requires the
template DNA, target sequence specific primers and mixture of dNTPs, and heat-stable
Taq DNA polymerase. A typical PCR involves repeated cycles of heating and cooling of
the reaction mixture to allow template DNA denaturation, primer annealing and new
DNA strand formation. Billions of amplicons will be generated at the end of 30-35
repetitive PCRcycles. At the end of 30 cycles, the specific sequence will be exponentially
amplified to generate multiple copies (230 copies) (amplicons).The basic PCR process has
been modified to expand its applications in various fields. PCR has been a widely used
technique as a diagnostic and research tool. The applications of PCR are progressively
growing and are used in many scientific disciplines including genetics, molecular
biology, forensic science, paternity testing, clinical diagnostics, microbiology,
environmental science, hereditary studies etc. Depending upon the application there may
be a variation in the PCR technique deployed which is another advantage of this method.
Random Primer ( )
5’ AAAAA 3’mRNA
Oligo(dT) Primer
5’ AAAAA 3’mRNA
PCR
Fig 3-3.2.2 A Representation of Real Time PCR Plot (Obtained from NCBI Probe)
a) Use of an intercalating dye like the SYBR® Green I dye which incorporates between
the base pairs of DNA. This detection method is suitable when the PCR reaction
generates a specific product, as the dye is capable of intercalating into any double-
stranded DNA product.
(The dye intercalates with double-stranded DNA in the reaction. In the bound state,
SYBR Green I exhibits 1000 fold more fluorescence than the unbound state. The
fluorescence signal increases in proportionwith the increase in amplified DNA)
PCR
Product
Hybridization
R Q
While in the case of TaqMan® probes, fluorescence occurs when the dye is clipped from
the probe during the polymerase extension.
R Q
Fig A
Q
Fig B
Scorpion primers are similar to Molecular Beacons™, but they contain a PCR
primer sequence. When the target DNA is amplified during PCR, the beacon fragment
binds to the newly synthesized DNA, and thus, separates the fluorophore from the
quencher. To prevent the stem-loop structure from being copied during PCR, a “PCR
blocker” is also incorporated in the hairpin. The intra-molecular binding between the
scorpion primer and the downstream PCR product is kinetically more favorable than that
of Molecular Beacons and TaqMan probes. Unlike the TaqMan and Molecular Beacons,
the Scorpion system do not require a separate probe and hence, gives a larger fluorescent
signal.
Target DNA
DNA Synthesis
Heat
Amplified DNA
Fig 3-3.2.3 Sequential Steps of Hot-Start PCR
Pick colony from a DNA library into a microcentrifuge tube or microtitre well.
Add PCR lysis Reagent, vortex, and heat for 5 min at 95-1000C.
Re-amplify
• The resultant product with known terminal sequences can now be used for
standard PCR.
Inverse PCR has many applications in molecular biology like the identification of
genomic inserts and the amplification and identification of sequences flanking
transposable elements.
Marker
In multiplex assays, false positive results are often obtained because each
amplified fragment provides an internal control for the other amplified fragments. It is
ideal for conserving costly polymerase and templates in short supply. Multiplex PCR can
be utilized to determine the amount of a particular template in a sample by exponential
amplification and internal standards. It is widely used in:
a) Pathogen identification,
c) Mutation analysis,
e) Template quantitation,
f) Linkage analysis,
g) RNA detection,
Fig 3-3.2.13 Comparison of symmetric PCR, standard asymmetric PCR, and LATE-PCR
Symmetric PCR reaches a plateau around 50 cycles and shows considerable variability in
kinetics with a simultaneous large end-point range. While, conventional asymmetric PCR
is flawed by delayed linear kinetics and lower end-point signals. Thus, LATE-PCR only
shows the efficiency in linear kinetics for over than 80 cycles.
Bibliography:
Innis MA, Myambo KB, Gelfand DH, Brow MA (1988). "DNA sequencing with
Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain
reaction-amplified DNA". Proc. Natl. Acad. Sci. U.S.A.85 (24): 9436–40.
doi:10.1073/pnas.85.24.9436.
Korbie DJ, Mattick JS (2008) Touchdown PCR for increased specificity and
sensitivity in PCR amplification. Nat Protoc3: 1452–1456.
Paul, N., Shum, J., Le, T. Hot Start PCR in Methods in Molecular Biology in RT-
PCR Protocols, Springer Science&Business Media, LLC 2010, 630: 301-18.
Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2006). "Thermostable DNA
Polymerases for a Wide Spectrum of Applications: Comparison of a Robust Hybrid
TopoTaq to other enzymes". In Kieleczawa J. DNA Sequencing II: Optimizing
Preparation and Cleanup. Jones and Bartlett. pp. 241–257. ISBN 0-7637-3383-0.
Sanchez JA, Pierce KE, Rice JE, Wangh LJ. Linear-after-the-exponential (LATE)-
PCR: an advanced method of asymmetric PCR and its uses in quantitative real-time
analysis. Proc Nat AcadSci USA 2004; 101:1933– 8.
Sandy B. Primrose & Robert W. Old; Principles of Gene Manipulation, 7th Edition.
Module 3 Lecture 4
3-4.2.1 Procedure:
1. The high-molecular-weight DNA strands are fractioned using restriction enzymes.
2. The DNA fragments are separated based on size by agarose gel electrophoresis.
3. The gel with the restricted fragments is then laid on a filter paper wick which serves as
a connection between the membrane and the high salt buffer.
4. The nitrocellulose membrane is placed on top of the gel and a tower of filter papers is
used to cover it and these are kept in place with a weight. The capillary action drives the
buffer soaking through the filter paper wick, through the gel and the membrane and into
the paper towels. Along with the buffer passing through the gel the DNA fragments are
also carried with it into the membrane and they bind to the membrane. This causes an
effective transfer of fragments (up to 15 kb in length taking around 18hours or overnight.
For DNA fragments larger than 15 kb, before blotting an acid such as diluted HCl is used
to treat the gel that depurinates the DNA fragments causing breakage of DNA into
smaller pieces, resulting in more efficient transfer from the gel to membrane.
Now a days blotting is also done by applying electric field. This electro blotting
technique depends upon current and transfer buffer solution to nucleic acids onto a
membrane. Following electrophoresis, a standard tank or semi-dry blotting transfer
system is set up. A stack is put together in the following order from cathode to anode:
sponge, three sheets of filter paper soaked in transfer buffer gel, PVDF or nitrocellulose
membrane, three sheets of filter paper soaked in transfer buffer and then again sponge.
Importantly the membrane should be located between the gel and the positively-charged
anode, as the current and sample will be moving in that direction. Once the stack is
prepared, it is placed in the transfer system, and suitable current is applied for a specific
period of time according to the materials being used.
5. For using alkaline transfer methods, the DNA gel is placed into an alkaline solution
(like that of sodium hydroxide) causing denaturation of the double-stranded DNA.
Denaturation in an alkaline environment enhances the binding between the negatively
charged DNA and the positively charged membrane, causing separation to single DNA
strands for further hybridization to the probe, alongside destroying any residual RNA that
may persist in DNA. The membrane is washed with buffer to remove unbound DNA
fragments.
7. The obtained membrane is then hybridized with a probe (a DNA fragment with a
specific sequence whose presence in the target DNA is to be determined).
8. Labeling of the probe DNA is done for easy detection, usually radioactivity is
incorporated or the molecule is tagged with a fluorescent or chromogenic dye. The
hybridization probe may be made of RNA, instead of DNA in some cases where the
target is RNA specific.
9. Washing of the excess probe from the membrane is done by using saline sodium
citrate(SSC buffer) after the hybridization step and the hybridization pattern is studied on
an X-ray film by autoradiography (for a radioactive or fluorescent probe), or via color
development on membrane if a chromogenic detection method is employed.
3-4.2 .3 Applications:
Southern blotting has been exploited for various applications which include:
3-4.3.1 Procedure:
The northern blotting involves the following steps:
1. Total RNA is extracted from a homogenized tissue sample or cells. Further eukaryotic
mRNA can then be isolated by using of oligo (dT) cellulose chromatography to isolate
only those RNAs by making use of a poly A tail.
2. The isolated RNA is then separated by gel electrophoresis.
3. The RNA samples separated on the basis of size are transferred to a nylon membrane
employing a capillary or vacuum based system for blotting.
4. Similar to Southern blotting, the membrane filter is revealed to a labeled DNA probe
that is complementary to the gene of interest and binds.
5. The labeled filter is then subjected to autoradiography for detection.
The net amount of a specific RNA in a sample can be estimated by using
Northern blot. This technique is widely used for comparing the amounts of a particular
mRNA in cells under different conditions. The separation of RNA samples is often done
on agarose gels containing formaldehyde as a denaturing agent as it limits the RNA to
form secondary structure.
The examination of the patterns of gene expressions obtained under given conditions
can help determine the function of that gene.
Northern blotting is also used for the analysis of alternate spliced products of same
gene or repetitive sequence motif by investigating the various sized RNA of the gene.
This is done when only probe type with variation in one location is used to bind to the
target RNA molecule.
Variations in size of a gene product may also help to identify deletions or errors in
transcript processing, by altering the probe target that can be used along the known
sequence and make it possible to determine the missing region of the RNA.
Incubate
Lyse bacteria,
neutralize,
Proteinase K, Wash
and Bake at 80 0C
It is a technique that employs a labeled complementary nucleotide strand (i.e. probe) for
localizing specific DNA or RNA sequence targets within fixed tissues and cells (i.e in
situ). Probes used for hybridization can be double-stranded DNA probes, single-stranded
DNA probes, RNA probes, synthetic oligonucleotides. There are two ways available to
detect DNA or RNA targets
(Obtained from
http://ajcp.ascpjournals.org/cont
ent/133/2/205/F1.expansion.htm
l)
FISH is a cytogenetic technique that uses fluorescent probes that bind to complementary
targets and sample is visualized using epi-fluorescence or confocal microscopy. Using
differently labeled probes, we can visualize several targets in a single sample. It is used
for spatial-temporal patterns of gene expression and resolving genetic elements in
chromosomal preparations.
Fig 3-4.6 Dots and slots in dot and slot blot hybridization
These procedures can only detect presence and absence of particular sequence or gene. It
cannot distinguish between two molecules of different sizes as they appear as single dot
on membrane. It also has application in detecting alleles that differ in single nucleotide
with the help of allele-specific oligonucleotides.
Bibliography
Sandy B. Primrose & Robert W. Old (2001); Principle of Gene Manipulation, 6th edition;
Blackwell Scientific Publ.
MODULE 3: LECTURE 5
PROBE AND TARGET SEQUENCES
3-5.1 Introduction
Probes are short section of DNA or RNA with an additional tagged or labeled chemical
entity that are used to bind to its complimentary strand and thereby allows detection of
candidate nucleic acid molecule. This chemically synthesized entity can be a fluorescent
molecule or it can be an attachment to a colored bead, or quantum dots (Cd-Se Qdots, Zn-
SQdots), photochromic compounds, isotopic labeling, non-isotopic labeling etc. It allows
us to visualize when a probe attaches to DNA, RNA or other target nucleic acids.
2. Target consists of template nucleic acid molecules with which probes will hybridize
and form complex and heterogeneous mixture.
The whole methodology revolves around the principle of hybridizing probe with a known
sequence to a target which may not have related sequences to anneal with. Conditions of
hybridization must be stringent enough to eliminate mismatched heteroduplexes. Low
concentration of salts and high temperature increases the stringent conditions. If the
probe used is small enough then hybridization reactions can be chosen such that
heteroduplex is unstable even when there is single mismatch.
(Homogenous
(Mixture of different Probe DNA
Target and labeled)
DNA fragments)
Denature Denature
Probe and target are first denatured and annealed forming the probe-probe homoduplexes
and target-target homoduplexes, but the desired reaction is probe-target heteroduplex
formation.
In vivo labeling: DNA and RNA can be directly labeled inside tissue culture cells by
adding labeled deoxynucleotides in culture plate in vivo. This method is restricted only to
prepare labeled viral DNA from virus-infected cells and to study RNA processing events.
a) Nick-translation
It involves insertion of random single-strand breaks called nicks in one of the strands of
double stranded target DNA which exposes 3′- OH termini and 5′-PO4termini.The nicks
are introduced by endonuclease like pancreatic deoxyribonuclease I (DNase I).
Addition of DNase I and multi-subunit enzyme E. coli DNA polymerase I is used for nick
translation which contributes both activities like:
(i) 5′ → 3′ exonuclease that attacks the exposed 5′ termini of a nick and sequentially
removes the nucleotides in 5′ → 3′ direction;
(ii) DNA polymerase adds nucleotides to the free 3′-OH group, in 5′ → 3′ direction
replacing the nucleotides removed by exonuclease and causing lateral displacement
(translation) of the nick.
This method requires 100-fold less radioactive precursor than in-vivo labeling method.
The amount of radiolabel incorporated depends on number of nicks created by DNase I.
Too much or too little nicking must be avoided. At low temperatures (about 15° C), the
disadvantage is that only one complete regeneration of existing nucleotide sequence takes
place and reaction does not proceed further.
The length of random primers is crucial. Primers shorter than 6 bases are very poor
primers, whereas those longer than 7 have higher tendency to self–anneal and cause
primer duplex. Thus, probe should be either 6 or 7 nucleotides in length.
Random priming is inherently simpler than nick translation because the requirements for
two nuclease activities are eliminated. The probes generated by random priming method
are more homogenous in size and behave more reproducibly in hybridization reactions.
Average size of probe cannot be controlled with great accuracy in nick translation
method.
Template DNA used preferably should not be closed, circular ds DNA because they are
inefficient templates. Shorter templates generate probes of low specific activity that may
not hybridize under stringent conditions.
The standard PCR reaction can be modified to incorporate labeled nucleotides. The
methods commonly used are:
Radiolabeled probes can be generated for both strands using equal concentrations of
primers or biased heavily in favor of one strand of DNA using higher concentration of
one primer.
5’ 3’
5’ 3’
3’ 5’
DNA Synthesis in presence of one
3’ 5’
5’ 3’
3’ 5’
5’ 3’
3’ 5’
The same methodology is used for labeling double stranded DNA. Fragments
which carry label at one end can only be generated using restriction enzyme which
cleaves at internal sites, thus producing two fragments which can be separated by gel
electrophoresis and purified. Fill-in end labeling is a popular approach to label large
DNA fragments using Klenow subunit of E. coli DNA polymerase.
The terminal nucleotide of blunt end DNA fragment can be replaced by weak 3’→5’
exonuclease and strong polymerase activity of klenow fragment.
Riboprobes are mostly created by run-off transcription in which RNA insert is cloned in
specialized plasmid vectors. The 5’ terminus of the transcript is fixed by bacteriophage
promoter, but 3’ terminus is defined by downstream site of cleavage by restriction
enzyme. Restriction enzymes that generate blunt or 5’-protuding termini produce best
linear templates. Enzymes generating protruding 3’ termini, should be avoided because
transcription of such templates results in synthesis of RNA molecules that are aberrantly
initiated at the termini of templates.
The DNA fragment to be transcribed can also be amplified in PCR with primers having
5’ ends encode synthetic promoters for bacteriophage RNA polymerases. Following
purification, the PCR products can be used as double stranded templates for in-vitro
transcription.
Fig 3-5.7.2.1 An example of fluorescein conjugated dUTP. The fluorescein group is linked to the 5′ carbon atom of the uridine
by a spacer group. Similarly, Rhodamine can also be used in place of fluorescein.
Biotin-Streptavidin Method: This method uses two ligands which has high affinity
towards each other: Biotin works as the reporter and the bacterial protein streptavidin is
used as the affinity molecule. Biotinylated nucleotides like bio-11-dUTP are used as
labeling agents with a spacer of 4-16 C atoms long between biotin and dNTP. However,
Biotin is a ubiquitous constituent of mammalian tissues and tends to stick easily to
certain type of nylon membranes which leads to high levels of background during in
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NPTEL – Biotechnology – Genetic Engineering & Applications
Chemiluminescence is the fastest and most sensitive assay using HRP (Horseradish
peroxidase) – luminal detection system. HRP catalyzes the oxidation of luminal in the
presence of H2O2, generating reactive peroxide that emits light at 425nm during
decomposition to its ground state.
BOOK
Samrook J and David W. Russell Molecular Cloning:A Laboratory Manual, 3rd Edition.
BIBLIOGRAPHY
Grunstein M., David S.,Hogness (1975). Colony hybridization: A method for the
isolation of clofied DNAs that contain a specific gene.Proc. Nat. Acad. Sci. USA Vol. 72,
No. 10, pp. 3961-3965.
BlouinJ. L., Rahmani Z., ChettouhZ., PrieurtM., FermaniantJ et al1990. Slot Blot Method
for the Quantification of DNA Sequences and Mapping of Chromosome Rearrangements:
Application to Chromosome 21.Am. J. Hum. Genet. 46:518-526.
MODULE 3- LECTURE 6
NUCLEIC ACID MUTAGENESIS: IN VIVO AND IN VITRO
3-6.1 Introduction
Mutation of the genes in higher organisms can be carried out by site- specific or
random variation in the gene expression cassette using the model microbes (eg. E.coli,
S.cerevisiae etc). The modification of the sequence of the gene is done by introducing an
alteration in the sequence. Mutagenesis is powerful genetic tool to study and characterize
functional elements of gene structure.
3-6.2 Classification
1) Site directed/ site specific mutagenesis- A specific site or stretch of some nucleic
acids is altered in a predetermined way by exploiting this approach. This is
accomplished by specific nucleic acid deletion or substitution to the targeted site
of mutation.
2) Mismatch mutagenesis- Mismatch mutagenesis is applied to create a desired point
mutation in a unique site precisely by introducing a mismatched nucleic acid base.
This method is generally used to determine the functional groups of a protein
encoded by that gene.
Several methods have been developed till now to obtain a successful site directed
mutation in the targeted site. Some of them are discussed below-
dUTP, resulting in increased level of dUTP in the cell. The uracil deglycosidase
deficiency prevents the removal of uracil from newly-synthesized DNA. As the double-
mutant E. coli replicates the phage DNA, its enzymatic machinery may therefore mis-
incorporate dUTP instead of dTTP, resulting in single stranded DNA which contains
ssUDNA. The ssUDNA is extracted from the bacteriophage released into the medium,
and used as a template for mutagenesis. An oligonucleotide containing the desired
mutation is used for primer extension. The heteroduplex DNA containing one parental
non-mutated strand of dUTP and a mutated strand containing dTTP is then transformed
into an E. coli strain carrying the wild type dut and ung genes. Here, the uracil-containing
parental DNA strand is degraded, so that nearly all resulting DNA consists of the mutated
strand.
A plasmid containing a target gene YFG (black segment) is cleaved with restriction
enzymes XbaI and BglII, each of which has unique restriction site in the entire
plasmid. The reaction mixture is separated by agarose gel electrophoresis. Two ss
oligonucleotides are synthesized by automated DNA synthesis. The sequences of the
oligonucleotides are complementary to each other and differ from wild-type
sequence at only a single position (black stripe) containing the desired changes. The
DNA is not entirely stable in aqueous solution. Under certain physiological conditions
glycosidic bond may be hydrolyzed impulsively and thousands of purine sites in DNA
are estimated to be depurinated daily in a cell. Several DNA repair pathways exist for the
DNA, however, if apurinic site is irrepairable, mis-incorporation of nucleotide may take
place during replication. Adenine is incorporated by DNA polymerases in an apurinic
site. Cytidine may also deaminated to uridine at lower rate of depurination and can form
G to A transition. Eukaryotic cells also contain 5’-methylcytosine, may be involved in
the control of gene transcription, which can become deaminated into thymine.
Alkylation and arylation of bases can cause errors in replication. Some alkylating agents
such as N-Nitrosamines may require the catalytic reaction of cytochrome-P450 for the
formation of a reactive alkyl cation. N7 and O6 of guanine and the N3 and N7 of adenine
are most susceptible to attack. N7 of guanine adducts form the bulk of DNA adducts, but
they appear to be non-mutagenic. Alkylation at O6 of guanine however is harmful
because excision repair of O6-adduct of guanine may be poor in some tissues such as the
brain. The O6 methylation of guanine can result in G to A transition, while O4-
methylthymine can be mis-paired with guanine. The type of the mutation generated
however may be dependent on the size and type of the adduct as well as the DNA
sequence. Ionizing radiations and reactive oxygen species often oxidize guanine to
produce 8-oxoguanine.
Some alkylating agents produce cross linking of DNA. Some natural occurring chemicals
such as psoralens after activation by UV radiation, and nitrous acid may also promote
cross linking. Inter-strand cross-linking is more lethal as it blocks replication and
transcription and can cause chromosomal breakages and rearrangements. Some cross
linkers such as cyclophosphamide, mitomycin C and cisplatin are used as anticancer
agent because of their high degree of toxicity to proliferating cells.
3-6.2.7 Dimerization
The planar structure of chemicals such as ethidium bromide and proflavine allows them
to insert between stacked bases in ds DNA. This insert causes the DNA's backbone to
stretch and makes slippage during replication more likely to occur since the bonding
between the strands is made less stable by the stretching. Forward slippage results in
deletion mutation, while reverse slippage results in an insertion mutation. The
intercalation of anthracyclines such as daunorubicin and doxorubicin also interferes with
the functioning of the enzyme topoisomerase II, blocking replication as well as causing
mitotic homologous recombination.
commonly used) and in Thale cress (Arabidopsis thaliana) and bacteria such as
Escherichia coli.
(Source: https://www.mdc-
berlin.de/8201913/en/research/research_teams/mobile_dna/research)
Figure [A] describes the components and structure of a two-component gene transfer
system based on Sleeping Beauty. A gene of interest (orange box) to be mobilized is
cloned between the terminal inverted repeats (IR/DR, black arrows) that contain binding
sites for the transposase (white arrows). The transposase gene (purple box) is physically
separated from the IR/DRs, and is expressed in cells from a suitable promoter (black
arrow). The transposase consists of an N-terminal DNA-binding domain, a nuclear
localization signal (NLS) and a catalytic domain characterized by the DDE signature.
Fig [B] illustrates the mechanism of Sleeping Beauty transposition system. The
transposable element carrying a gene of interest (GOI, orange box) is maintained and
delivered as part of a DNA vector (blue DNA). The transposase (purple circle) binds to
its sites within the transposon inverted repeats (black arrows). Excision takes place in a
synaptic complex. Excision separates the transposon from the donor DNA, and the
double-strand DNA breaks that are generated during this process are repaired by host
factors. The excised element integrates into a TA site in the target DNA (green DNA)
that will be duplicated and will be flanking the newly integrated transposon.
Bibliography
Rashtchian A,Thornton C.G. and Heidecker G. 1992. A novel method for site directed
mutagenesis using PCR and uracil DNA glycosylase; Genome Res., 2: 124-130.
Stanford et al.2001. Gene-trap mutagenesis: past, present and beyond. Nature Reviews
Genetics 2, 756-768.
Storici F, Resnick MA. 2006. The delitto perfetto approach to in vivo site-directed
mutagenesis and chromosome rearrangements with synthetic oligonucleotides in yeast.
Methods Enzymol.; 409: 329-45.