Molecular Probes and Ligase Chain reaction

Promila SheoranPh.D. BiotechnologyGJU S&T Hisar

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.

THEORY OF LCR

•The principle of LCR is based in part on the ligation of two adjacent syntheticoligonucleotide 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.

•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.

•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 LCRprimers 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.

CURRENT APPLICATIONS OF LCR

LCR assays have been developed for the detection of genetic diseases as wellas 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

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.

•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.

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.

•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.

•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.

•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.

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.

Thank You