Presentation on Linkers &Adapters used in rDNA Technology

Presentation on Linkers &Adapters used in rDNA Technology

1. Linkers & Adapters Dr.V.Malathi,Associate Professor in Biochemistry,Ethiraj College for Women,Chennai-8

2. 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. Dr.V.Malathi,Associate Professor in Biochemistry,Ethiraj College for Women,Chennai-8

3. 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. Dr.V.Malathi,Associate Professor in Biochemistry,Ethiraj College for Women,Chennai-8

4. Dr.V.Malathi,Associate Professor in Biochemistry,Ethiraj College for Women,Chennai-8

5. Dr.V.Malathi,Associate Professor in Biochemistry,Ethiraj College for Women,Chennai-8

6. https://biocyclopedia.com/index/biotechnology/genes_genetic_engineering/techniques_of_genetic_engineering/biotech_insertion_dna_fragment_vector.php Dr.V.Malathi,Associate Professor in Biochemistry,Ethiraj College for Women,Chennai-8

7. 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. Dr.V.Malathi,Associate Professor in Biochemistry,Ethiraj College for Women,Chennai-8

8. Dr.V.Malathi,Associate Professor in Biochemistry,Ethiraj College for Women,Chennai-8 https://biocyclopedia.com/index/genetics/genetic_engineering_and_biotechnology_recombinant_dna/blunt_end_ligation_by_t4_dna_ligase.php

9. 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. Dr.V.Malathi,Associate Professor in Biochemistry,Ethiraj College for Women,Chennai-8

10. Dr.V.Malathi,Associate Professor in Biochemistry,Ethiraj College for Women,Chennai-8 Image source: www.slidesharenet/dr.sandeepaims/gene-cloning-sandeep-jan-2016

11. 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. Dr.V.Malathi,Associate Professor in Biochemistry,Ethiraj College for Women,Chennai-8

12. Dr.V.Malathi,Associate Professor in Biochemistry,Ethiraj College for Women,Chennai-8 http://ecoursesonline.iasri.res.in/Courses/Principles%20of%20Plant%20Biotechnology/GPBR311/Data%20Files/lec19.pdf

13. 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 . Dr.V.Malathi,Associate Professor in Biochemistry,Ethiraj College for Women,Chennai-8

14. Dr.V.Malathi,Associate Professor in Biochemistry,Ethiraj College for Women,Chennai-8 http://ecoursesonline.iasri.res.in/Courses/Principles%20of%20Plant%20Biotechnology/GPBR311/Data%20Files/lec19.pdf

15. 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. Dr.V.Malathi,Associate Professor in Biochemistry,Ethiraj College for Women,Chennai-8

16. Dr.V.Malathi,Associate Professor in Biochemistry,Ethiraj College for Women,Chennai-8 http://ecoursesonline.iasri.res.in/Courses/Principles%20of%20Plant%20Biotechnology/GPBR311/Data%20Files/lec19.pdf

17. 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. Dr.V.Malathi,Associate Professor in Biochemistry,Ethiraj College for Women,Chennai-8

18. 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.pdf http://biosiva.50webs.org/dnacloning.htm http://ecoursesonline.iasri.res.in/Courses/Principles%20of%20Plant%20Biotechnology/GPBR311/Data%20Files/lec19.pdf Dr.V.Malathi,Associate Professor in Biochemistry,Ethiraj College for Women,Chennai-8