Presentation on Linkers &Adapters used in rDNA Technology

Dr.V.Malathi,Associate Professor in Biochemistry,Ethiraj College
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Linkers & Adapters

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Adaptor/ linker
• An adapter or a linker in genetic engineering is a
• short,
• chemically synthesized,
• single-stranded or double-stranded oligonucleotide
• that can be ligated to the ends of other DNA or RNA molecules.
• It may be used to add sticky ends to cDNA allowing it to be ligated
into the plasmid much more efficiently.

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Linkers
• Linkers are the chemically synthesized double stranded DNA oligonucleotides
• They containing one or more restriction sites
• These can be cleaved by restriction enzymes, e.g. Eco RI, Hind III, Bam HI,
etc.
• Linkers are ligated to blunt end DNA by using DNA ligase.
• Both the vector and DNA are treated with restriction enzyme to develop
sticky ends.
• The staggered cuts i.e. sticky ends are then ligated with T4 DNA ligase with
very high efficiency to the termini of the vector and recombinant plasmid
DNA molecules are produced.
• Linkers are supplied as phosphorylated and non-phosphorylated forms.

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https://biocyclopedia.com/index/
biotechnology/genes_genetic_engineering/
techniques_of_genetic_engineering/
biotech_insertion_dna_fragment_vector.php

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• Linkers are synthesized as single stranded oligos with the restriction site in
the middle and designed to have a palindrome sequence. These single
strands self-ligate to form a double strand.
• Disadvantage
• When linkers added to link at the end of blunt end of gene interest, then
there is an possibility of joining of multiple linkers at the end.
• This makes some time larger genes and waste of linker molecules. This
problem is overcome by using adapters.

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https://biocyclopedia.com/index/genetics/
genetic_engineering_and_biotechnology_recombinant_dna/
blunt_end_ligation_by_t4_dna_ligase.php

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Adapters
• Adapter is a synthetic, double stranded oligonucleotide
• used to attach sticky ends to a blunt ended molecule.
• It contain normal 5' and 3' end at blunt end and
• the sticky end of adapter molecule is modified in such manner that it contain OH group on both 5' and
3' ends.
• This is achieved by using alkaline phosphatases.
• In contrast to linkers, adapters contain preformed sticky ends and joining blunt ends.
• Because of lack of 5' phosphate group on sticky end prevents adapter polymer formation.
• After the adaptors have been attached the abnormal 5'OH terminus is converted to the natural 5'P form
by treatment with the enzyme polynucleotide kinase, producing sticky ended fragment that can be
inserted into an appropriate vector.
• The adaptor molecules are synthetic deoxynucleotides that can be used to join two incompatible
cohesive ends, two blunt ends or a combination of both.
• Such adaptors are of several types viz., preformed, conversion and single stranded adaptors.

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Image source: www.slidesharenet/dr.sandeepaims/gene-cloning-sandeep-jan-
2016

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Preformed adaptors
• Preformed adaptors are short DNA duplexes with at least one cohesive end.
• The problem of internal cleavage of the insert DNA can be overcome by using
a preformed adaptor that will introduce a new restriction site.
• For example, an adaptor having BamHI cohesive ends and sites HpaII and
SmaI can be attached to passenger DNA and inserted into a BamHI in vector.
• After cloning, passenger DNA can be excised from the hybrid by using any one
of the enzymes that recognize the restriction sites within the adaptor region.

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http://ecoursesonline.iasri.res.in/Courses/Principles%20of%20Plant%20Biotechnology/GPBR311/Data%20Files/lec19.pdf

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Conversion adaptors
• Conversion adaptors are synthetic oligonucleotides bearing different cohesive
restriction termini.
• Such adaptors enable vector molecules that have been cleaved with one
endonuclease to be joined to passenger fragments that have been cleaved
with another.
• Often these adaptors contain internal restriction sites that permit recovery of
the passenger fragment, for example, the EcoRI-BamHI adaptor contains a
site for XhoI.

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Single stranded adaptors
• Single stranded adaptors can be used to make 3’-protruding cohesive ends
compatible with 5’ protruding ends.
• Such adaptors permit the insertion of passenger fragments into sites on vectors
from which they would otherwise be precluded because of incompatible
cohesive ends.

for Women,Chennai-8
http://ecoursesonline.iasri.res.in/Courses/Principles%20of%20Plant%20Biotechnology/GPBR311/Da
ta%20Files/lec19.pdf

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Adaptors Vs Linkers
• The choice of use of either a linker or an adaptor is based on the application
and the ends of the DNA of interest that needs to be altered as it does not
have the desired restriction site at the end nor has that site internally that will
digest the DNA to several fragments.
• Preference should be given to ligation using cohesive ends as these are ligated
more efficiently than blunt ends, i.e. use adaptors over linkers.

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References
• https://unacademy.com/lesson/linkers-and-adaptors/Y1IVX4CD
• http://www.genelink.com/Literature/ps/Adaptors_COA_Ver5.2.pdf
• https://nptel.ac.in/content/storage2/courses/102103013/pdf/mod2.p
df
• http://biosiva.50webs.org/dnacloning.htm
• http://ecoursesonline.iasri.res.in/Courses/Principles%20of%20Plant%
20Biotechnology/GPBR311/Data%20Files/lec19.pdf

Presentation on Linkers &Adapters used in rDNA Technology

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