第二章基因工程的“工具”.ppt

Enzymes are the Tools of DNA Technology The tools of DNA technology are the same enzymes used by cells to modify their own DNA: DNA Polymerases DNA Ligase One special class of enzyme is pivotal to the cloning of DNA and many other techniques used in DNA Technology These enzymes are the restriction endonucleases Restriction - Because for the way they work, they restrict bacteriophages to only one host bacterial strain. They are also restricted to acting on only specific DNA sequences Endonuclease - They cut nucleic acids in the middle not just the ends Restriction Endonucleases There are a number of different sub classes of restriction endonucleases Type I - Recognize specific sequences and cut DNA at a nonspecific site than 1,000 bp away Type II - Recognize palindromic sequences and cut within the palindrome Type III - Recognize specific 5-7 bp sequences and cut 24-27 bp down stream of the site. Type II restriction endonucleases are the most useful class as they recognize specific palindomic sequences in DNA and cut the sugar phosphate backbone within the palindrome What is a Palindrome? A palindrome is anything that reads the same forwards and backwards: English palindromes: Mom Dad DNA Palindromes Because DNA is double stranded and the strands run antiparallel, palindromes are defined as any double stranded DNA in which reading 5’ to 3’ both are the same Some examples: 5）Uses of Restriction Endonucleases Because restriction endonucleases cut specific sequences they can be used to make “DNA fingerprints” of different samples of DNA. As long as the cutting site changes on the DNA or the distance between cutting sites changes, fragments of different sizes will be made. Because Type II restriction endonucleases cut only at palindromes, they leave “sticky ends” that will base pair with any other fragment of DNA cut with the same enzyme. This is useful in cloning. R. E.s and DNA Ligase Can be used to make recombinant DNA Some Commonly Used Restriction Enzymes E