Tools for the gene technologist

Restriction enzymes

• restriction endonucleases are a class of enzymes from bacteria which recognise and break down the DNA of invading viruses known as bacteriophages (phages for short)
• bacteria make enzymes that cut phage DNA into smaller pieces
• these enzymes cut the sugar-phosphate backbone of DNA at specific places within the molecule
  • this is why they are known as endonucleases ('endo' means within)
• their role in bacteria is to restrict a viral infection, hence the name restriction endonuclease or restriction enzyme

Each restriction enzyme binds to a specific target site on DNA and cuts at that site

• bacterial DNA is protectedd from such attack either by chemical markers or by not having target sites
• these target sites/restriction sites are specific sequences of bases
  • for example, BamHI always cuts DNA where there is a GGATCC sequence on one strand and its complementary sequence, CCTAGG, on the other
    
  • this sequence reads the same in both directions: it is palindromic
• many, but not all, restriction sites are palindromic
• restriction enzymes either cut straight across the sugar-phosphate backbone to give blunt ends or they cut in a staggered fashion to give sticky ends
• sticky ends are short lengths of unpaired bases
• they are known as sticky ends because they can easily form hydrogen bonds with complementary sequences of bases on other pieces of DNA cut with same restriction enzyme
• when long pieces of DNA are cut there will be a mixture of different lengths
• to find the specific piece of DNA required involves separating the lengths of DNA using gel electrophoresis and using gene probes
• multiple copies of the required piece of DNA can be made using the polymerase chain reaction (PCR)

Restriction enzymes are named by an abbreviation which indicates their origin

• roman numbers are added to distinguish different enzymes from the same source

Because many proteins have been sequenced, it is possible to use the genetic code to synthesise DNA artificially from nucleotides rather than cutting it out of chromosomal DNA or making it by reverse transcription

• genes and even complete genomes, can be made directly from DNA nucleotides without need for template DNA
• scientists can do this by choosing codons for the amino acid sequence they need
• the sequence of nucleotides is held in a computer that directs the synthesis of short fragments of DNA
• these fragments are then joined to make a longer sequence of nucleotides that can be inserted into plasmids for use in genetic engineering
• this metod is used to generate novel genes that are used, for example, in the synthesis of vaccines and they have even been used to produce the genomes of bacteria consisting of a million base pairs

Vectors

Inserting a gene into a plasmid vector

In order to get a new gene into a recipient cell, a go-between called a vector often has to be used and one type of vector is a plasmid

• these are small circular pieces of double-stranded DNA
• they occur naturally in bacteria and often contain genes for antibiotic resistance
• …