Oligonucleotide ligation assay description with colony hybridization

1. PRESENTED BY:
Syeda Tamanna Yasmin
M.Sc. Microbiology ,ADBU

2.  A Blotting technique is a method of transferring
proteins, DNA or RNA onto a carrier-
Nitrocellulose or Polyvinylidene fluoride or Nylon
membrane.
 Hybridization is a technique that measures degree
of similarity between pools of DNA, RNA or
proteins sequences.
 Colony hybridization (blot) is a method of
identifying those bacterial colonies in a plate
which contains a specific DNA sequence or gene
of interest.
 Developed by Grunstein and Hogness in 1975.

5. Stringnent hybridization conditions can be
used to ensure that only such sequences that
are identical to the probe are identified.
Less stringnent hybridization conditions can be
used to identify both identical and related
sequences.
Probes corresponding to a conserved functional
domain of a gene can be used to identify
members of a gene family.

6. Colonies whose DNA-prints exhibit
hybridization can be picked from the reference
plate.
 In this method, they isolated clones of ColE1
hybrid plasmids that contain Drosophila
melanogaster genes for 18 and 28S rRNAs.

7.  Investigated for potential in determining populations of specific
gene sequences in environmental samples.
 Cross-hybridizations among two degradative plasmids, TOL and
NAH, and two cloning vehicles, PLAFR1 and RSF1010, were
determined. This allowed detection of one colony containing TOL
plasmid among 10 (6) E.coli colonies of nonhomologous DNA.
 Findings indicated that standard sole carbon source enumeration
procedures for degradative populations lead to overestimations due
to non-specific growth of other bacteria on the microcontaminant
carbon sources present in the media.
 It also estimates of substrates mineralization rates and past
exposure to environmental samples and hope eventually allow
enumeration of any specific gene sequences in environment-
anabolic & catabolic genes. (Help in recombinant DNA clones)

8.  Commonly known as the OLA,
the Oligonucleotide Ligation Assay is an analyte
specific reagent (ASR) that is centered around the
hybridization of a PCR primer with an exact
match to a target sequence.
 The oligonucleotide ligation assay is a genotypic
assay for the detection of resistance-associated
mutations to reverse transcriptase and protease
inhibitors in human immunodeficiency virus type
1 subtype B..

9.  The target DNA sequence is PCR-amplified prior
to the ligation step of the annealed probes:
 One of the detection probes consists of a sequence
complementary to the target sequence .
 The second detection probe is fully
complementary to the target sequence and serves
as a reporter.
 Detection probes bind next to each other
 The product is captured by anti-TAG on the
microsphere surface.

10. The OLA consists of two
phases:
a multiplex PCR
amplification and
a multiplex OLA, in a single-
tube format.

11.  In the first reaction a PCR primer is hybridized to
the target sequence. The primers are designed with
either the normal or mutant nucleotide(s) at the 3'
end and a tail of different lengths to distinguish
various PCR products based on size at the 5' end. .
 The second reaction is a ligation reaction. A
common primer contains a fluorescent dye marker
at the 3' end and meets the first primer right over
the nucleotide position that will be altered in a
mutant allele.

12. The hybridization of oligonucleotide .
The need for these primers to directly adjacent
to one another.
The requirement that the oligonucleotide have
two bases perfectly complementary.

18. Advantages :
Rapid
Easy
single-tube analysis
high throughput capability on capillary
sequencer
genotyping and automation capability.
Disadvantages :
Requirement for an automated sequencer.