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Restriction mapping
Restriction mapping is a technique that generates a map of DNA molecules by identifying sites for restriction enzymes through data from restriction digests. The process involves the use of restriction endonucleases and gel electrophoresis, which enables visualization of DNA fragments. Historically, the discovery of restriction enzymes has been significant in molecular biology, leading to their application in DNA sequencing and plasmid engineering.
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Introduction to restriction mapping, a method for illustrating DNA sites for restriction enzymes.
Description of techniques like restriction endonucleases and gel electrophoresis for DNA mapping.
Overview of the historical development and key discoveries related to restriction enzymes.
Information about commonly used restriction enzymes, their recognition sites, and palindromic nature.
Steps for restriction mapping: isolation of DNA, digestion with enzymes, and electrophoresis.
Usage of agarose gels for visualizing DNA fragments after restriction digestion and electrophoresis.
Details on the electrophoresis power supply used for separating DNA fragments.
Explanation of how gel results are visualized under UV light.
Steps in constructing a restriction map using reciprocal and double digests.
Information on DNA length and the cleavage process using two restriction enzymes.
Discussion of reciprocal and double digestion techniques used in restriction mapping.
Representation of restriction maps showing a linear arrangement of cleavage sites.
Importance of restriction maps in molecular biology for DNA engineering and sequencing.
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Restriction mapping
- 1.
- 2. What is restriction mapping? Itis a process of generating a map of a DNA molecule either linear or circular( a plasmid),indicating where the sites for certain restriction enzymes are using the data from restriction digests with those enzymes.
- 3. TECHNIQUES INVOLVED : USE OF RESTRICTION ENDONUCLEASES GEL ELECTROPHORESIS
- 4. What are restrictionendonucleases (REs)? How can REs be used to identify DNA molecules?
- 5. Historical accounts ofrestriction enzymes 1. The term restriction enzyme originated from the studies on phage lambda(bacterial virus) bacteriophage. 2. In 1950s Salvador Luria and Giuseppe Bertani found that bacteriophage can grow well in E.coli. 3. In 1960s Werner Arber and Matthew Messelson found that restriction is caused by enzymatic cleavage of phage DNA(type 1=EcoRl). 4. In 1970s Hamilton O.Smith and Thomas Kelly discovered Hind II restriction enzyme. 5. Daniel Nathans and Kathleen showed the cleavage of DNA by restriction enzymes into fragments by using gel electrophoresis. Thus this showed that restriction enzyme can be used in mapping. 6. In 1978 Werner Arber,Daniel Nathans,Hamilton O.Smith were awarded the Nobel prize for the discovery of restriction enzymes.
- 6. REs with 6-nucleotiderecognition sites (6-cutters) are widely used in molecular biology RE Strain of origin Recognition site EcoRI E. coli (strain RY13) G AA T T C Hind III H. influenza AA G C T T Recognition sites are often palindromes
- 7. GAATTC CTTAAG 3’ 5’ 5’ 3’ GAATTC CTTAAG 3’ 5’ 5’ 3’ G AATTC CTTAA G 3’ 5’ 5’ 3’ EcoRIrecognition site is a palindrome with an axis of symmetry EcoRI dimer binds sequence and catalyzes double-strand cleavage Products have “sticky ends”: unpaired hydrogen bonds on nitrogen bases
- 8. METHOD OF RESTRICTION MAPPING 1.ISOLATION OF DNA 2. DIGESTION WITH A SPECIFIC RESTRICTION ENZYME. 3. EXTRACTION OF DNAAND ELECTROPHORESIS.
- 9. Agarose Gels Tovisualize the results of a restriction digest, you need to separate the different fragments of DNA, and determine their size We will do this by agarose gel electophoresis
- 10. Agarose Agarose isvery water soluble polysaccharide Forms porous, aqueous gels after heating and cooling
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- 12. Gel Visualized UnderUV Light
- 13. CONSTRUCTION OF ARESTRICTION MAP 1. It involves successive digests with 2 individual enzymes , where we extract each fragment produced in the individual digest with either enzyme A or enzyme B and then cleave it with other enzymes. 2. The original DNA sample is also digested by a mixture of both the enzymes to confirm the results of individual successive digests. 3. The former and the latter are known as Reciprocal digests and Double digest respectively. 4. Using the information we can find the over lapping regions in A and B digest and find out the sites of cleavage by A and B .This will then allow us to prepare a restriction map.
- 14. CLEAVAGE OF DNABY 2 RESTRICTION ENDONUCLEASES AAND B LENGTH OF EXPERIMENTAL DNA = 5000 bp
- 15. TECHNIQUE OF RECIPROCAL ANDDOUBLE DIGESTS
- 17. Restriction mapin the form of linear arrangement of cleavage sites.
- 18. SIGNIFICANCE 1. In molecularbiology restriction maps are used as a reference to engineer plasmid or other relatively short pieces of DNA. 2. It is used to sequence the whole molecule of DNA and run it through computer program that will find the recognition sites that are present for every restriction enzyme known.