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Ligase Chain Reaction(LCR)
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
Ligase Chain Reaction(LCR)
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
Ligase Chain Reaction(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.
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.
Ligase Chain Reaction(LCR)
Ligase Chain Reaction(LCR)
Separation And
      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.
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).




                                                    25
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.

                                                     26
ELISA




CRACKJRF.COM
Add enzyme labeled (alkaline
 phosphatase) Antidigoxigenin
         antibodies




           CRACKJRF.COM
Add substrate (PNP
 phosphate)




    CRACKJRF.COM
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


                                                     30
NONISOTOPIC DETECTION
        (By Zebala And Barany)

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.

                                                 31
THE DETECTION OF THE PRODUCTS
          FROM G-LCR
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.
                                                   32
Ligase Chain Reaction(LCR)
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.
Ligase Chain Reaction(LCR)
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.
Ligase Chain Reaction(LCR)
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

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Ligase Chain Reaction(LCR)

  • 2. PRESENTED BY: • HIRA BATOOL • ALEENA NAYYAR • NOOR-UL-AIN YASIN • FARHAN-UL-HAQ • SANA BANGASH
  • 3. 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.
  • 4. 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
  • 5. 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.
  • 6. 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.
  • 7. PRINCIPLE • The absence of the ligated product therefore indicates at least a single base-pair change in the target sequence
  • 9. REQUIREMENTS • Template DNA. • Polymerase enzymes. • dNTPs. • Reaction Buffer. • Thermo-cycler. • DNA probes. • Radioactive Tags and/or flourescent dyes.
  • 10. 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.
  • 11. 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.
  • 12. 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)
  • 13. IMPORTANT FACTORS Accurate results from LCR assays depend on a variety of factors, including: Primer design  Reaction Conditions
  • 14. 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
  • 16. 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).
  • 17. 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.
  • 18. 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.
  • 19. 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.
  • 20. • 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.
  • 21. 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.
  • 24. Separation And 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.
  • 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). 25
  • 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. 26
  • 28. Add enzyme labeled (alkaline phosphatase) Antidigoxigenin antibodies CRACKJRF.COM
  • 29. Add substrate (PNP phosphate) CRACKJRF.COM
  • 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 30
  • 31. NONISOTOPIC DETECTION (By Zebala And Barany) 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. 31
  • 32. THE DETECTION OF THE PRODUCTS FROM G-LCR 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. 32
  • 34. 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.
  • 35. 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.
  • 36. APPLICATIONS • Infectious diseases can be detected easily. • Enhanced detection of Phytophthora infestans. • Early Diagnosis of Tuberculosis Meningitis
  • 37. 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 .
  • 38. 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.
  • 39. 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.
  • 41. 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.
  • 42. 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.
  • 44. 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