Restriction fragment

Fig. 1. Southern blot analysis of DNA showing (a) step 1, an agarose gel containing separated restriction fragments of DNA, denoted by (—), which is immersed in NaOH to denature the double-stranded stmcture of DNA, and then transferred by capillary flow to a nitrocellulose filter. In step 2, the bound DNA is allowed to hybridize to a labeled nucleic acid probe, and the unbound probe is washed off In step 3, the filter is placed into contact with x-ray film resulting in (b) bands of exposure on the film which are detected after development and correspond to regions where the restriction fragment is...

Linker-Scanning Mutagenesis. Using linker-scanning mutagenesis (24) small sequences of DNA are removed and replaced with a synthetic restriction fragment or linker. This technique is commonly used in analysis of promoters and other control sequences in DNA, while preserving the spatial relationship between the sequences. 

Therefore, DNA restriction fragments having such sticky ends can be joined together to create new combinations of DNA sequence. If the fragments are derived from DNA molecules of different origin, novel recombinant forms of DNA are created. 

An restriction fragment of foreign DNA can be inserted into a plasmid having an... 

I A small piece of DNA called a primer, whose sequence is complementary to that on the 3 end of the restriction fragment 1 The four 2 -deoxvribonucleoside triphosphates (dNTPs)... 

When reaction is complete, the product consists of a mixture of DNA fragments of all possible lengths, each terminated by one of the four dye-labeled dideoxyribonucleotides. This product mixture is then separated according to the size of the pieces by gel electrophoresis (Section 26-2), and the identity of the terminal dideoxyribonucleotide in each piece—and thus the sequence of the restriction fragment—is identified simply by noting the color with which the attached dye fluoresces. Figure 28.8 shows a typical result. 

Figure 28.8 The sequence of a restriction fragment determined by the Sanger dideoxy method can be read simply by noting the colors of the dye attached to each of the various terminal nucleotides.

Hamet, P., Kong, D., Pravenec, M., Kunes, J., Kren, V., Klir, P., Sun, Y.L., Tremblay, J. (1992). Restriction fragment length polymorphism of hsp70 gene, localized in the RTl complex, is associated with hypertension in spontaneously hypertensive rats. Hypertension 19,611-614. 

Fig. 1. Electrophoretic mobility of Hind lll-restrict fragments of X DNA after UV irradiation (A) and DNA electrophoregram trace (B) 1 - control, 2 - after UV irradiation for 3 hours (3.64 J/m2 ), 3, 4 - the same in the presence of Q-AR in concentrations of ICHM and lO M, 5, 6 - the same in the presence of Q-AR at concentrations of lO M and lO M. The arrow shows...

DEMONSTRATION OF LINKAGE TO AN R F L P (restriction fragment iength polymorphism)... 

Restriction endonucleases facilitate diagnosis of genetic diseases by revealing restriction fragment length polymorphisms. 

A similar analysis could be made for a number of other diseases. Point mutations are usually defined by sequencing the gene in question, though occasionally, if the mutation destroys or creates a restriction enzyme site, the technique of restriction fragment analysis can be used to pinpoint the lesion. Deletions or insertions of DNA larger than 50 bp can often be detected by the Southern blotting procedure. 

The differences in DNA sequence cited above can result in variations of restriction sites and thus in the length of restriction fragments. An inherited difference in the pattern of restriction (eg, a DNA variation occurring in more than 1% of the general population) is known as a restriction fragment length polymorphism,... 

Abbreviations STS, sequence tagged site RFLP, restriction fragment iinked poiymorphism SNP, singie nucieotide poiymorphism YAC, yeast artificiai chromosome BAC, bacteriai artificiai chromosome PCR, poiymerase chain reaction. 

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