Restriction endonucleases cleavage sites

Fig. 1. Nucleotide sequence of the SFV 26 S RNA (top row), the corresponding amino acid sequence (middle row), and the amino acid sequence of the Sindbis virus structural proteins (bottom row). Nucleotides are numbered from the 5 end of the RNA molecule and all amino adds from the amino terminus of each protein. The amino- and the carboxyl-terminal ends of each protein are indicated hy arrows, glycosylation sites by triangles, and membrane-spanning regions of the viral glycoproteins by underlines for Sindbis virus and overlines for SFV. Amino acids in boxes are negatively charged (Asp and Glu), and those circled are positively charged (Lys and Arg). Some restriction endonuclease cleavage sites are shown on the nucleotide sequence. The alignment of the amino acid...

The MCS is a stretch of unique restriction endonuclease cleavage sites between the promoter and the poly (A) signal in expression plasmid vectors, and is used as a common area to insert a cDNA or gene of interest. By using the MCS, the genes of interest can be inserted and assayed with ease. Sticky-end and blunt-end vector strands are available at different restriction sites. Although the MCS is very convenient, it should be noted that these sites usually remain in the mRNA transcribed from the expression unit of the vector and may reduce its stability or the efficiency of translation. Furthermore, one must be careful not to produce an ATG codon at the junctional sequence between the MCS and the insert, because it may work as a false start codon in the resulting mRNA and interfere with the production of the correct product. 

Galibert et al. (1974) to deduce the sequence of a 48 nucleotide long fragment from X174. While its use for routine analysis has now been superceded problems can still arise for which the newer procedures are not entirely satisfactory. Confirming the sequence of chemically synthesized oligonucleotides for use as primers and the analysis of short sequences adjacent to restriction endonuclease cleavage sites are conveniently carried out by these methods. 

Gronenbom, B. and J. Messing, 1978. Methylation of single-stranded DNA in vitro introduces new restriction endonuclease cleavage sites. Nature 272, 375. 

The array of restriction endonuclease cleavage sites which has been inserted near the N-terminus of the /3-galactosidase gene occupies positions 6231 (EcoRI site) to 6238 (centre of the HaeIII site) cf. page 134, chapter 4). A restriction map of the closely related M13mp8 is given in Appendix 8. 

Fig. 3. Universal primers for PCR amplification of endogenous retroviral reverse transcriptase. The primers anneal within the retroviral reverse transcriptase gene at the position indicated by the thick lines in Fig. 2. The degenerate primer pair has been used successfully,21 but it gives a number of nonspecific products. Substitution of inosine at some of the degenerate positions increases the effective concentration of primer. Xba and coRI restriction endonuclease cleavage sites are underlined.

The most widely used genetic screening technique, PCR-RFLP detects a mutation at a specific restriction endonuclease cleavage site at the mutation locus [9], The products from other techniques such as ARMS (amplification refractory mutation system), SSCP (single-strand conformational polymorphism), HPA (heteroduplex polymorphism), and CDCE (constant denaturant capillary electrophoresis) and PCR are usually separable using polymer networks. 

For a generation of transgenic mice, it is not desirable to micro-inject vector sequences. The PI and BAC vectors are equipped with several rare restriction endonuclease cleavage sites flanking the DNA insert. Thus, the DNA insert frequently can be cleaved from the vector with these enzymes. 

Templeton A (1983) Phylogenetic inference from restriction endonuclease cleavage site maps with particular reference to the humans and apes. Evolution 37 221-244... 

Improvement (i) came with the development of phage (e.g. M13 and its constructs such as M13mpl9), plasmid (e.g. the pUC series) and phage-mid (e.g. pBluescript II KS+/-) DNAs with high copy numt is, resistance to specific antibiotics, multiple restriction endonuclease cleavage sites and known coding sequences into which the DNA to be sequenced could be inserted with precision. 

The top part of the figure schematically illustrates the immune interferon gene containing unique restriction endonuclease cleavage site. The remainder of the figure outlines the synthetic strategy as discussed in the text. 

Fig. 1 Some restriction endonuclease cleavage sites in SV40 DNA.

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