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INTRODUCTION• A method of DNA amplification similar to PCR.• LCR amplifies the probe molecule rather than producing amplicon through polymerization of nucleotides.• Two probes are used per each DNA strand and are ligated together to form a single probe.• LCR uses both a DNA polymerase enzyme and a DNA ligase enzyme to drive the reaction.
OBJECTIVESLCR in PCR world?????Describe the ligase chain reaction and highlight its qualitiesin light of its use as a diagnostic detection methodHow it allows the discrimination of DNA sequences differingin only a single base pairAdvantages and Applications of LCR
PRINCIPLE• The principle of LCR is based four oligonucleotides, two adjacent oligo-nucleotides which uniquely hybridize to one strand of target DNA and a complementary set of adjacent oligonucleotides, which hybridize to the opposite strand• 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.
PRINCIPLE• 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.• If there is a mismatch at the primer junction, it will be discriminated against b y thermostable ligase and the primers will not be ligated.
PRINCIPLE• The absence of the ligated product therefore indicates at least a single base-pair change in the target sequence
THERMAL CYCLERA thermocycler is an expensive laboratory apparatus used to amplify DNA under controlled temperature conditions. In thermal cycler repeated temperature changes result in the separation of the ligated (bound) units from the target.
DNA PROBES• 4 oligonucleotide probes are required.• Probes are designed to match two adjacent sequences of specific target DNA .• The probes are attached to radioactive substances or tagged with a dye facilitating easy detection of the target sequence.
LIGASE & POLYMEASE• LCR uses thermostable DNA ligase to amplify the allele-specific product.• Purified from an E. coli strain containing the cloned ligase gene from Thermus aquaticus• Taq DNA Ligase is active at elevated temperatures (45°C-65°C)• Taq DNA polymerase (same as in PCR)
IMPORTANT FACTORSAccurate results from LCR assays depend ona variety of factors, including: Primer design Reaction Conditions
DESIGN OF PROBES• LCR probes w ith a single base-pair overhang, rather than blunt ends, should be used.• This minimizes target-independent ligation. Template
Three steps are:• Denaturation: Heat double-stranded DNA to denature it usually at 950C for several minutes.• Annealing: Annealing of probes to target DNA ( at 600C).• Ligation: Joining of the probes by thermostable DNA ligase. ( at 600C).
STEP 1: DENATURATION• DNA is subjected to heat, that causes its separation into single-stranded nucleic acid.Denaturing of the initial double-stranded DNA sample.
STEP 2: Annealing of probes to Target DNA• Two sets of probes are designed to anneal at a specific region of the sample DNA.• This begins the target DNA production.• Each probe pair is hybridized to adjacent positions on the template.
STEP 3: Ligation• The gap created by the joining of two probes is recognized by the enzyme DNA ligase and is ligated and creates a continuous DNA sequence that is used to identify the presence of the target molecule.• DNA-ligase will only ligate primers that have perfectly annealed to the sample DNA.
• The mixture is then heated so that the probe and target DNA are separated.• Again cool, this repeated temperature changes result in the separation of the ligated units from the target.• The separated ligated unit becomes the target for the next cycle or round of ligation.
CONTD.• Each cycle results in a doubling of the target nucleic acid molecule.• By repeating the above steps through several cycles, there is a rapid exponential accumulation of the specific target fragment of DNA.
Separation And DetectionSeparation: Gel electrophoresis is used for the separation of the amplified LCR products. The target molecule is analyzed on a polyAcrylamide gel electrophoresis (PAGE).Detection: Autoradiography is used to detect the LCR products. It is a technique where the probes are labeled with radioactive molecules, which on exposure to X-rays can be visualized.
LIGASE-MEDIATED DNA DETECTIONThe separation of LCR products and primers canbe achieved by denaturing gel electrophoresis,and the LCR product is detected byAUTORADIOGRAPHY NONISOTOPIC DETECTION METHODUsing fluorescently labeled primers, detection ofthe LCR product can also be accomplished usinga fluorescent DNA sequencer in conjunction witha GENESCANNER (applied biosystems). 25
NON ISOTOPIC DETECTIONIt may either be analyzed using electrophoresis orELISANonisotopic detection uses one digoxigenin-labeledprimer; the LCR products are detected in a southern blotformat after gel electrophoretic separation.One lcr primer of a pair is labeled with biotin at the 5end, whereas the other primer is labeled with anonisotopic reporter at the 3 end.Reporter groups tested so far include a fluoresceindye in blue (FAM, 5-carboxyfluorescein) and digoxigenin. 26
DIRECT DETECTION OF FAM-LABELED LCR PRODUCTSFluorometry showed poor sensitivity, whereas the useof digoxigenin reporter in conjunction with anti-digoxigenin antibodies coupled to alkaline phosphatase(AP) greatly improved the sensitivity. Subsequentdetection of the AP could be achieved using colorimetric,fluorescent, or luminogenic substrates.Winn- deen et al. reported that the luminogenicsubstrate lumiphos 530 gave the highest sensitivity in amicrotiter plate assay. This sensitivity was only 10-foldless than with detection methods using radioisotopes ora fluorescent DNA sequencer 30
NONISOTOPIC DETECTION (By Zebala And Barany)They utilized primer pairs in which one primerwas labeled with a poly(dA) tail at the 5 endwhereas the 3 end of the other primer wastagged with biotin.The ligated products were captured from thesolution via hybridization of their poly(dA) tailswith poly(dT)-coated paramagnetic iron beads andsubsequent magnetic separation.Only the captured LCR products will carry a S-coupled biotin molecule, which can be detectedwith a streptavidin-AP conjugate and acolorimetric substrate. 31
THE DETECTION OF THE PRODUCTS FROM G-LCRRadioactively labeled nucleotides were used tofill in the gap between the primers, so that the G-LCR products can be detected by autoradiographyafter gel electrophoresis. Alternatively, the primerscan be end labeled with radioisotopes.Nonisotopic detection of G-LCR products can beachieved by using pairs of primers labeled withbiotin or fluorescein, respectively. Ligated oligonucleotides were captured onantifluorescein-coated micro particles anddetected with an antibiotin-AP conjugate. AP activity was subsequently detected with thefluorescent substrate methylumbelliferonephosphate. 32
AIMS & APPLICATIONS• It aims to amplify oilgonucleotide probes or primers specific for short DNA target sequences.• Nucleotide amplification has made this technique more specific and sensitive.• There are several applications of this technique. some of them are mentioned here.• point mutation detection based on a ligase chain reaction. This method has two advantages:(i) use of Cleavage increases the accuracy of ligation(ii) (ii) a template independent ligation does not occur in LCR due to a special design of primers.
APPLICATIONS• For eg.The LCR Chlamydia trachomatis (urinogential infection)test is a highly sensitive nonculture technique.• The LCR has been used for genotyping studies to detect tumors and identify the presence of specific genetic disorders such as sickle cell disease caused by known nucleotide changes.
APPLICATIONS• Infectious diseases can be detected easily.• Enhanced detection of Phytophthora infestans.• Early Diagnosis of Tuberculosis Meningitis
ADVANTAGESLCR-based systems have some advantages over the PCR-based amplification systems• Misincorporated nucleotides are not replicated in the product allowing amplification of a different sequence than that found in the target nucleic acid.• The LCR reactions are also more specific for the nucleotide allowing for higher discriminatory power against mismatches at a single chosen site .
ADVANTAGES• LCR is very useful for determining the nucleotide at a specific site such as a single base change, e.g., single-nucleotide polymorphisms (SNPs) used in mapping complex genomes.• The LCR cycle has only two short steps allowing for shorter amplification times.• The usually small target of LCR, 36 to 60 nucleotides, does not require high- quality large fragment nucleic acids.
Continue…• The commercial LCR kit, the Abbott LCx System is less affected by inhibitors in some specimens.• Large numbers of samples can be analyzed by LCR faster than with culture-based methods.• A simple and sensitive miRNA assay was developed with ligase chain reaction (LCR) based on specific ligation of DNA probes by using miRNAs as the templates.
Continue….• LCR have the additional advantage that it do not require viable organisms in a specimen• A single specimen can be used to detect multiple different pathogens, provided suitable primers are available.• Easily obtained specimens such as urine can be used for diagnostic purposes, making screening of large numbers of persons practical.
DRAW BACKS• One problem with LCR is that the target is amplified, resulting in a contamination risk.• Phosphate inhibits the ligase chain reaction when it is added before the amplification stage.• Variation in copy number for the plasmid containing the LCR target is also a potential source of error.
REFRENCES• M Wiedmann, W J Wilson, J Czajka, et al. 1994, Ligase chain reaction (LCR)-- overview and applications. Genome Res, 3: S51-S64• F Barany,1991, The ligase chain reaction in a PCR world. Genome Reserch,doi:10.1101/gr.1.1.5• http://nar.oxfordjournals.org/content/23/4/675• http://groups.molbiosci.northwestern.edu/holmgren/Glossary/Definitions/Def- L/ligase_chain_reaction.html• http://momsorganicmarket.com/ns/DisplayMonograph.asp?StoreID=a6b40ae98c7 842a98fc8de4784880288&D ocID=genomic-ligasechainreaction• https://docs.google.com/viewer?a=v&q=cache:RglokYrT44wJ:www.biotechniques.c om/multimedia/archive/00036/BTN_A_04373ST03_O_36971a.pdf+conditions+of+l igase+chain+reaction+requirements&hl=en&pid=bl&srcid=ADGEESi4WxRKtSD5fr95 p8tNBr2_l8-JOuRDv7- VTFl49EceV3K4VLplvOauBM7bWZ6iizVbl0n0o7fS_0f4njpzHxr_D7kQvHgCBa12N5TI XY3uZoQcFizEBDmRPvagvF9Y-ujPs8Lh&sig=AHIEtbTi- E9fKMV9_NqgbFnFTY88vVl7Dw• http://momsorganicmarket.com/ns/DisplayMonograph.asp?StoreID=a6b40ae98c7 842a98fc8de4784880288&DocID=genomic-ligasechainreaction