Recombinant DNA Technology

1. Molecular Probes and
Ligase Chain reaction
Promila Sheoran
Ph.D. Biotechnology
GJU S&T Hisar

2. Ligase Chain Reaction
•LCR evolved as a very promising diagnostic technique that is often utilized in
conjunction with a primary PCR amplification. LCR employs a thermostable ligase and
allows the discrimination of DNA sequences differing in only a single base pair.
• The power of LCR is its compatibility with other replication-based amplification
methods. By combining LCR with a primary amplification, one effectively lines up the
crosshairs to distinguish single base-pair changes with pinpoint accuracy.

3. THEORY OF LCR
•The principle of LCR is based in part on the ligation of two adjacent synthetic
oligonucleotide primers, which uniquely hybridize to one strand of the target DNA.
• The junction of the two primers is usually positioned so that the nucleotide at the 3'
end of the upstream primer coincides with a potential single base-pair difference in the
targeted sequence.
•This single base-pair difference may define two different alleles, species, or other
polymorphisms correlated to a given phenotype.
• If the target nucleotide at that site complements the nucleotide at the 3' end of the
upstream primer, the two adjoining primers can be covalently joined by the ligase.

4. •The unique feature of LCR is a second pair of primers, almost entirely
complementary to the first pair, that are designed with the nucleotide at the 3' end
of the upstream primer denoting the sequence difference.
• In a cycling reaction, using a thermostable DNA ligase, both ligated products can
then serve as templates for the next reaction cycle, leading to an exponential
amplification process analogous to PCR amplification.
•If there is a mismatch at the primer junction, it will be discriminated against by the
thermostable ligase and the primers will not be ligated.
• The absence of the ligated product therefore indicates at least a single base pair
change in the targer sequence.

5. •The example shown is an
LCR with matched target (L.
monocytogenes) and
mismatched target (L.
innocua). The pathogenic
bacteria L. monocytogenes
can be distinguished from
other closely related Listeria
spp. (e.g., L. innocua) by a
single base-pair difference
in the 16S rDNA.
• L. monocytogenes has an
A-T base pair at nucleotide
1258, whereas L. innocua
has a G-C base pair at this
position. (Top) DNA is
denatured at 94°C and the
four LCR
primers anneal to their
complementary strands at
65°C which is approximately
5°C below their Tm.
•Thermostable ligase will
only ligate primers that
are perfectly
complementary to their
target sequence and
hybridize directly adjacent
to each other (as shown
with L. monocytogenes,
left).
•Primers that have at least
a single base-pair
mismatch at the 3' end
contributing to the
junction of the two
primers will not ligate (as
shown with L. innocua,
right). The discriminating
primers have a 2-bp
noncomplementary AA
tail at their 5'
ends to avoid ligation of
the 3' ends.

6. CURRENT APPLICATIONS OF LCR
LCR assays have been developed for the detection of genetic diseases as well
as for the detection of bacteria and viruses.
1) Detection of Genetic Diseases
2) Detection of Bacterial Pathogens
3) Detection of Viruses
4) Detection of Other Target Sequences

7. Molecular Probes
•Molecular probes are small DNA segments (genomic DNA, cDNA or synthetic
oligonucleotides) or RNA segments (often synthesized on DNA template) that
recognize complementary sequences in DNA or RNA molecules and thus allow
identification and isolation of these specific DNA sequences from an organism.
• Antibodies are also occasionally use as probes to recognize specific protein
sequences.

8. •DNA/RNA probe assays are faster and sensitive, so that many conventional
diagnostic tests for viruses and bacteria involving culturing of the organism, are
being fast replaced by antibody and DNA probe assays.
•While culture tests can take days or even months, molecular probe assays can be
performed within few hours or minutes.
•Therefore, the use of DNA probes has become today's most sophisticated and
sensitive technology for a variety of uses involving biological systems both in basic
and applied studies including their commercial use.

9. Labelling of probes
•The detection of homologous sequences after hybridization with the probe is like
finding a needle in the haystack.
• Therefore, for the success of DNA probe assay, it is necessary to develop simple,
safe and sensitive techniques for their use.
•As probes transmit no signal of their own, they have to be (i) labelled either with
radioactive isotopes (e.g. 32P dCTP) or with non-radioactive signal molecules
(e.g. biotin or digoxigenin) without impairing the hybridization ability of these
probes.
•These signal molecules may include fluorescent antibodies and enzymes that
produce colour changes in dyes and chemiluminescent catalyst.

11. •After hybridization with radioactively labelled probe, hybrids are detected by
autoradiography.
•32P has the advantage over other radioisotopes, since it has high specific activity.
However, in general, radioisotopes have some disadvantages.
•They are difficult to handle and expensive to dispose off. Detection by
autoradiography, while sensitive, may take a long time if there are few counts in the
hybrid.
• Furthermore, radioisotopes have a short half life (e.g. 32P has a half-life of 14.3
days) and therefore experiments should be completed preferably within one half-
life.

13. •Recent advances in nucleic acid technology now offer alternatives to radioactively
labelled probes.
•One procedure that is becoming increasingly popular is biotin labelling of nucleic
acids.
• After hybridization and washing, detection of hybrids is done by a series of
cytochemical reactions which finally gives a blue colour whose intensity is
proportional to the amount of biotin in the hybrid.
•There are several advantages of using biotinylated probes. For example, these assays
employ non-toxic materials, whose half-life is longer.
•These probes can be prepared in advance in bulk and stored at - 20°C for repeated
uses.

14. •Digoxigenin is another chemical derived from plants and used for non- radioactive
labelling of probes.
•An antibody associated with an enzyme (anti- digoxigenin-alkaline phosphatase
conjugate) is used for the detection of the presence of digoxigenin.
•The probe may be labelled with digoxigenin-11-dUTP supplied with a digoxigenin kit.
The labelled and denatured probe may be used for hybridization with denatured DNA
on Southern blots or for in-situ hybridization.
• After hybridization the membrane or the slide may be washed and the membrane or
the slide is transferred into detection buffer containing 20 μg/ml of anti-digoxigenin
fluorescein and 5% (w/v) BSA (bovine serum albumin).
•The system is incubated for 1h at 37°C, and then the membrane/slide is washed in
detection buffer three times (8 min each at 37°C). Alkaline phosphatase activity can be
detected using BCIP (5-bromo 4-chloro 3-indolyl phosphate) and NBL (nitroblue
tetrazolium) as dye substrate.

15. Applications of molecular probes
Molecular probes can be used for both basic and applied studies in the field of molecular
biology and biotechnology. These uses, include the following:
(i) 'Restriction fragment length polymorphisms (RFLPs)' and their uses for preparation
of molecular maps and other purposes.
(ii) Screening of genomic and cDNA libraries for isolation of gene's etc.
(iii) Location of genes or DNA sequences on chromosomes, through in situ hybridization
or ISH
(iv) Use in diagnosis of diseases and DNA fingerprinting.

16. Thank You