Restriction enzymes recognition sequence

Shankarappa B, Vijayananda K, Ehrlich G. 1992. Silmut a computer program for the identification of regions suitable for silent mutagenesis to introduce restriction enzyme recognition sequences. Biotechniques 12 882. 

Denature template DNA and anneal primers that contain a specific restriction-enzyme recognition sequence at the 5 end... 

For amplification, use 25 cycles of denaturation at 95°C for 30 s, annealing at 60°C (50°C for the first 4-5 cycles if using 3 primers with heterologous restriction enzyme recognition sequences at their 5 ends) for 1.5 mm and extension at 72°C for 3 min The last cycle should be followed by a final extension at 72°C for 15 min After completion, the reaction can be stored frozen or, for a short time, at4 C... 

These are used to search for short sequences such as restriction enzyme recognition sites. The main difference between them is in the form of their output. 

Unfortunately, many SNPs do not lead to an alteration of a restriction enzyme recognition site in the genome sequence. For these SNPs, alternative approaches have been developed that use PCR and gel electrophoresis. In one approach, one... 

Figure 5.7-8. Schematic overview of PCR-RFLP. Double-stranded DNA molecules are shown for DNA sample A and B. These two samples are PCR amplicons from genomic DNA. The two sequences differ for the base pair highlighted in red. Sample A contains a restriction enzyme recognition site for EcoRl (underlined), and sample B does not. After restriction enzyme digestion, agarose gel electrophoresis results in different DNA fragments for the two samples. (This figure is available in full color at ftp //ftp.wiley.com/public/ sci tech med/pharmaceutical biotech/.)...

We discuss these two modules together because both allow you to search a nucleotide sequence for positions where an arbitrary subsequence may be silently added—that is, the nucleotide sequence will be modified in place without altering its translation. The Restriction Site Addition is just a special case of the Short Sequence Addition where all the arbitrary subsequences are restriction enzyme recognition sites. 

Just as you may wish to add arbitrary subsequence to a gene, you may want to remove them. The Subtraction modules use a variant of the least different RSCU algorithm (see Subheading 3.4) to disrupt target sequences with as few synonymous codon substitutions as possible. The Restriction Site Subtraction module is just a specialized case of Short Sequence Subtraction, in which all arbitrary subsequences are restriction enzyme recognition sites. Unlike the Addition modules, where you can select which subsequences to add, the Subtraction modules attempt to remove every instance of target subsequence. 

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