restriction enzymes 350 Home R. Ward: Spring 2001

Transcription

1 restriction enzymes 350 Home Restriction Enzymes (endonucleases): molecular scissors that cut DNA Properties of widely used Type II restriction enzymes: recognize a single sequence of bases in dsdna, usually symetrical (palindromic) cleave both strands, generally within the recognition sequences leaving blunt or sticky ends leave a 3'-hydroxyl on one side of the cut and a 5'-phosphate on the other side of each strand 1

2 Examples of restriction enzymes 350 Home 2

3 Palindromic sequences 5 NNNNNNNNNGGATCCNNNNNNNNNN 3 3 NNNNNNNNNCCTAGGNNNNNNNNNN 5 Axis of symmetry 5 3 G C G C A T T A 350 Home v 3

4 350 Home Gene cloning = replication of a target sequence of DNA insert target sequence into an easily replicated vector insert the vector into a single bacteria (transformation) allow the bacteria to amplify vector has sequences that enable coordinated replication of the recombinant vector DNA DNA Cloning: Vectors Properties of useful vectors: Cloning vectors cloning Vector DNA can be introduced into a bacteria Vector contains a replication origin so it can replicate inside a host cell vector contains antibiotic resistance gene or other selectable marker gene that allows easy identification of transformed bacterial colonies Plasmid: insert DNA max size= 15 kb; autonomous replication; contains antibiotic resistance genes Bacteriophage lambda: insert = 25 kb; recombinant DNA packaged into phage particles used to infect E. coli BACs: bacteria artificial chromosomes insert size kb YACs: yeast articifical chromosomes kb (1 mb) generate fragments with a restriction enzyme. Sources can be whole DNA sample (genomic), or DNA generated from RNA of particular tissue mix with linearized (restricted) plasmid (cut with same enzyme ligate get two products: plasmid with insert, plasmid only have site in middle of gene whose function is disrupted upon insertion bacterial amplification transform bacteria with plasmid/insert construct grow (plate) single cell dervied colonies on media carrying selectable agent (antiotic, usually) grow single colony derived bacterial strains that survive the antibiotic and exhibit evidence of insertion maintain as colony on plate or as frozen solution. Library is a collection of clones 4

5 350 Home Cloning vectors cloning Plasmid: insert DNA max size= 15 kb; autonomous replication; contains antibiotic resistance genes Bacteriophage lambda: insert = 25 kb; recombinant DNA packaged into phage particles used to infect E. coli BACs: bacteria artificial chromosomes insert size kb YACs: yeast articifical chromosomes kb (1 mb) generate fragments with a restriction enzyme. Sources can be whole DNA sample (genomic), or DNA generated from RNA of particular tissue mix with linearized (restricted) plasmid (cut with same enzyme ligate get two products: plasmid with insert, plasmid only have site in middle of gene whose function is disrupted upon insertion transform bacteria with plasmid/insert construct grow (plate) single cell dervied colonies on media carrying selectable agent (antiotic, usually) grow single colony derived bacterial strains that survive the antibiotic and exhibit evidence of insertion maintain as colony on plate or as frozen solution. Library is a collection of clones 5

6 350 Home 6

7 350 Home 7

8 350 Home Main point on BACs and YACs is the large size insert they can carry 8

9 350 Home Purifying insert involves separating plasmid from bacterial chromosomal DNA, followed by restriction with original cloning enzyme, then separation of plasmid (or other vector) from insert Methods of separation include electrophoresis You can also use PCR to amplify the insert directly from bacterial colonies or purified vectors by using the DNA sequence info flanking the vector cloning site to design forward and reverse primers. 9

10 Libraries: Collections of DNA clones 350 Home Genomic libraries are made from genomic DNA directly from the plant s DNA cdna libraries are made from DNA fragments that are created with reverse transcriptase from mrna isolated from the plant Complete libraries hypothetically contain one or more copies of every sequence from the source tissue. Libraries maintained as bacterial or yeast colonies 10

11 Subcloning 350 Home Lambda (Phage) sub-libraries of individual YAC or BAC inserts enable manipulation of 20kb or so subsequences from that YAC/BAC insert Each sub-library can then be sub-cloned further into plasmids for even higher resolution. 11

12 cdna 350 Home Big message here is that mrna can be converted into DNA, called cdna cdna inserts do not have promoter or introns from the original genomic locus that the mrna came from 12