RESTRICTION MAPPING

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RESTRICTION MAPPING

RESTRICTION MAPPING : 

RESTRICTION MAPPING Restriction mapping is a method used to map an unknown segment of DNA by breaking it into pieces and then identifying the locations of the breakpoints. This method relies upon the use of proteins called restriction enzymes, which can cut, or digest, DNA molecules at short, specific sequences called restriction sites. After a DNA segment has been digested using a restriction enzyme, the resulting fragments can be examined using a laboratory method called gel electrophoresis, which is used to separate pieces of DNA according to their size. Restriction maps help to know which RE(Restriction enzyme) acts upon each allele.

METHOD FOR CONSTRUCTING A RESTRICTION MAP : 

METHOD FOR CONSTRUCTING A RESTRICTION MAP It involves digesting the unknown DNA sample in three ways. Here, two portions of the DNA sample are individually digested with different restriction enzymes, and a third portion of the DNA sample is double-digested with both restriction enzymes at the same time. Next, each digestion sample is separated using gel electrophoresis, and the sizes of the DNA fragments are recorded. The total length of the fragments in each digestion will be equal. However, because the length of each individual DNA fragment depends upon the positions of its restriction sites, each restriction site can be mapped according to the lengths of the fragments The final drawing of the DNA segment that shows the positions of the restriction sites is called a restriction map

Restriction site; : 

Restriction site; Restriction sites, or restriction recognition sites, are specific sequences of nucleotides that are recognized by restriction enzymes. The sites are generally palindromic, (because restriction enzymes usually bind as homodimers) and a particular restriction enzyme may cut the sequence between two nucleotides within its recognition site, or somewhere nearby. For example, the common restriction enzyme EcoRI recognizes the palindromic sequence GAATTC and cuts between the G and the A on both the top and bottom strands.

Restriction enzymes: : 

Restriction enzymes: Restriction enzymes are enzymes that cut DNA at specific recognition sequences called "sites." Restriction enzymes are endonucleases that recognize specific 4 to 8 base regions of DNA. For example, one restriction enzyme, Eco RI, recognizes the following six base sequence: 5' . . . G-A-A-T-T-C . . . 3' 3' . . . C-T-T-A-A-G . . . 5' A piece of DNA incubated with Eco RI in the proper buffer conditions will be cut wherever this sequence appears. As the site is palindromic; each strand of the DNA can self-anneal and the DNA forms a small cruciform structure:

Restriction Mapping: : 

Restriction Mapping: The combination of all restriction site in a specific DNA sequence is called restriction mapping. Because it indicate every single site in the sequence , that can be recognised and cut by a specific enzyme. The distance between restriction enzyme sites can be determined by the patterns of fragments that are produced by the restriction enzyme digestion This gives the information about the structure of unknown piece of DNA

Restriction enzymes : : 

Restriction enzymes :

For example: : 

For example: Take a pBluescript portion of plasmid (3kb) with a known; Restriction site (Eco RI &Bam HI) Inserted of 2kilobases long

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Digest plasmid with an enzyme that you know is in the pBluescript plasmid. For example, you know that there is only one Bam HI site in pBluescript, and it is in the multiple cloning site next to the Eco RI site If you digest this plasmid with Bam HI, there are two possibilities: 1) There are no Bam HI sites in the insert. If this is the case, when you run this digestion on a gel you will see only one DNA fragment, and it will be 5.0 kb long (3.0 kb of pBluescript DNA and 2.0 kb of insert DNA). 2) There is a Bam HI site in the insert. If this is the case, then the enzyme will cut the circular plasmid in two places, in the pBluescript part of the plasmid and in the insert.

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In 2nd case, you will end up with two fragments of DNA. One will be pBluescript with some of the insert still attached and the other will be just insert. The sizes of the two fragments (determined by electrophoresis) will tell you where the site is. These two possibilities are shown below

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In the second case, where there is a site in the insert, the gel might look like this:

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This case,show two pieces of information: That there is a Bam HI site in the insert, where the site is in relation to the one end of the insert. When the Bam HI digestion is separated on an agarose gel, the sizes of the two fragments can be determined. In the above gel, the fragments are 3.6 kb and 1.4 kb. Therefore, we know that the Bam HI site is 1.4 kb away from the right hand side of the insert . In this way, you have "mapped" the Bam HI site: By testing the insert for the presence and location of sites of many different restriction enzymes,a "restriction map" of the clone is made.

COMPARING SEQUENCE OF TWO DIFFERENT NUCLEOTIDE SEQUENCE : : 

COMPARING SEQUENCE OF TWO DIFFERENT NUCLEOTIDE SEQUENCE : In this we take enzyme specific for one nucleotide Enzyme specific for SeqA not for SeqB, so it cut the SeqA nucleotide only. Tested:- Agarose gel electrophoresis where SeqA &SeqB specified by their segment SeqA-Smaller; SeqB-Larger Smaller the size faster it run on gel and settle at bottom In this method, DNA fragment get separated based on their molecular weight & it motivated by electric current and settle on gel. In this activated electrode are at either end producing electric field and gel with slots made on it.

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In slot DNA + Enzyme is put As DNA is a charged molecule due to presence of phosphate group DNA move towards positive pole and separated based on electrode which determine how the molecule move across the gel Larger/heavier fragment move towards positive It can be visualize by adding enzyme Ethidium bromide + DNA -------------> Red orange color bands Methyline blue+ DNA---------------- Blue color bands

Uses of Restriction Mapping: : 

Uses of Restriction Mapping: It cut a large piece of DNA into smaller fragments to allow it to be sequenced. It is an easy way to compare DNA fragments without having any information of their nucleotide sequence. We can detect fragment size generated by knowing ; Original size before cutting it with enzyme Specific spot where the enzyme has acted For example: we can isolate two clones for a gene that are 8 kb and 10 kb long, they overlap as they have sequences in common. A restriction map can tell you how much they overlap It can tell which parts of the two clones are identical and which parts are different.

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