LIGASE CHAIN

REACTION

PRESENTED BY:

• HIRA BATOOL• ALEENA NAYYAR• NOOR-UL-AIN YASIN• FARHAN-UL-HAQ• SANA BANGASH

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.

OBJECTIVES

LCR in PCR world?????

Describe the ligase chain reaction and highlight its qualities in light of its use as a diagnostic detection method

How it allows the discrimination of DNA sequences differing in only a single base pair

Advantages 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

REQUIREMENTS & FACTORS

REQUIREMENTS

• Template DNA.• Polymerase enzymes.• dNTPs.• Reaction Buffer.• Thermo-cycler.• DNA probes. • Radioactive Tags and/or flourescent

dyes.

THERMAL CYCLER A 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 FACTORS

Accurate results from LCR assays depend on a 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

STEPS AND PHASES OF

LCR

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.

• 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 2: Annealing of probes to Target DNA

• 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.

STEP 3: Ligation

• 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.

• 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.

CONTD.

ANALYSIS & DETECTION

Separation: 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.

Separation And Detection

25

LIGASE-MEDIATED DNA DETECTION

The separation of LCR products and primers can be achieved by denaturing gel electrophoresis, and the LCR product is detected by AUTORADIOGRAPHY

NONISOTOPIC DETECTION METHOD Using fluorescently labeled primers, detection of the LCR product can also be accomplished using a fluorescent DNA sequencer in conjunction with a GENESCANNER (applied biosystems).

26

NON ISOTOPIC DETECTION

It may either be analyzed using electrophoresis or ELISA Nonisotopic detection uses one digoxigenin-labeled primer; the LCR products are detected in a southern blot format after gel electrophoretic separation.

One lcr primer of a pair is labeled with biotin at the 5' end, whereas the other primer is labeled with a nonisotopic reporter at the 3' end.

Reporter groups tested so far include a fluorescein dye in blue (FAM, 5-carboxyfluorescein) and digoxigenin.

CRACKJRF.COM

ELISASA

C-G

B

D

SA

C

B

CRACKJRF.COM

Add enzyme labeled (alkaline phosphatase) Antidigoxigenin antibodies

SA

C-G

B

D

SA

C

B

CRACKJRF.COM

Add substrate (PNP phosphate)

SA

C-G

B

D

SA

C

B

30

DIRECT DETECTION OF FAM-LABELED LCR PRODUCTS

Fluorometry showed poor sensitivity, whereas the use of digoxigenin reporter in conjunction with anti-digoxigenin antibodies coupled to alkaline phosphatase (AP) greatly improved the sensitivity. Subsequent detection of the AP could be achieved using colorimetric, fluorescent, or luminogenic substrates.

Winn- deen et al. reported that the luminogenic substrate lumiphos 530 gave the highest sensitivity in a microtiter plate assay. This sensitivity was only 10-fold less than with detection methods using radioisotopes or a fluorescent DNA sequencer

31

They utilized primer pairs in which one primer was labeled with a poly(dA) tail at the 5' end whereas the 3' end of the other primer was tagged with biotin.

The ligated products were captured from the solution via hybridization of their poly(dA) tails with poly(dT)-coated paramagnetic iron beads and subsequent magnetic separation.

Only the captured LCR products will carry a S'-coupled biotin molecule, which can be detected with a streptavidin-AP conjugate and a colorimetric substrate.

NONISOTOPIC DETECTION

(By Zebala And Barany)

32

Radioactively labeled nucleotides were used to fill in the gap between the primers, so that the G-LCR products can be detected by autoradiography after gel electrophoresis. Alternatively, the primers can be end labeled with radioisotopes.

Nonisotopic detection of G-LCR products can be achieved by using pairs of primers labeled with biotin or fluorescein, respectively.

Ligated oligonucleotides were captured on antifluorescein-coated micro particles and detected with an antibiotin-AP conjugate.

AP activity was subsequently detected with the fluorescent substrate methylumbelliferone phosphate.

THE DETECTION OF THE PRODUCTS FROM G-LCR

APPLICATIONS,ADVANTAGES & DRAWBACKS

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.

ADVANTAGES

LCR-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=a6b40ae98c7842a98fc8de4784880288&D ocID=genomic-ligasechainreaction

• https://docs.google.com/viewer?a=v&q=cache:RglokYrT44wJ:www.biotechniques.com/multimedia/archive/00036/BTN_A_04373ST03_O_36971a.pdf+conditions+of+ligase+chain+reaction+requirements&hl=en&pid=bl&srcid=ADGEESi4WxRKtSD5fr95p8tNBr2_l8-JOuRDv7-VTFl49EceV3K4VLplvOauBM7bWZ6iizVbl0n0o7fS_0f4njpzHxr_D7kQvHgCBa12N5TIXY3uZoQcFizEBDmRPvagvF9Y-ujPs8Lh&sig=AHIEtbTi-E9fKMV9_NqgbFnFTY88vVl7Dw

• http://momsorganicmarket.com/ns/DisplayMonograph.asp?StoreID=a6b40ae98c7842a98fc8de4784880288&DocID=genomic-ligasechainreaction