The document summarizes the ligase chain reaction (LCR) technique. LCR amplifies DNA sequences using four probes and ligase and polymerase enzymes instead of amplifying DNA through nucleotide polymerization like PCR. It can detect single nucleotide polymorphisms. The process involves denaturing the DNA, annealing probes to the target sequence, and ligating the probes if they match, repeating for exponential amplification. Products are detected through gel electrophoresis or non-radioactive methods. LCR has applications in infectious disease detection, genotyping and mutation analysis.

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

27. ELISA
CRACKJRF.COM

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