BIOL 311 Human Genetics Recombinant DNA Methods Restriction Enzymes Cloning Vectors Reading Chap 5 pp 129 133 139 146 Lecture Outline 1 Restriction enzymes 2 Conventional cloning vectors and applications 3 Cloning vectors for genomics Lecture 1 Restriction enzymes a Discovered in bacteria and blue green algae b Enzymes that recognize and cleave specific sites on DNA Are endonucleases cleave within a DNA duplex c Function to protect organism from virus infections the host s DNA is typically modified by methylation to prevent restriction enzymes from damaging host DNA d Useful type for recombinant DNA known as type II restriction enzymes e Most of the commonly used restriction enzymes recognize 4 6 bp palindromic restriction sites Rare cutters such as Not I and Sfi I recognize larger 8 bp sites and are useful for genome mapping f May produce 5 overhangs 3 overhangs or blunt ends g Uses of restriction enzymes Create fragments for cloning recombinant DNA create fragments for end labeling used in restriction mapping genomic maps RFLP analysis Examples EcoRI Escherichia coli R factor 5 G AATT C3 3 C TTAA G5 5 overhang PstI Providencia stuarti 5 C TGCA G3 3 G ACGT C5 3 overhang AluI Arthrobacter luteus 5 AG CT3 3 TC GA5 Blunt end Naming 1st letter is genus name 2nd and 3rd letter is species name numbered in order of discovery Small scale restriction digests 20 ul reaction 2 ul of 10x restriction buffer buffer Mg2 salts 1 10 ul of DNA sample 0 5 1 ug 1 ul 1 2 units restriction enzyme 1 Each restriction enzyme unique use a special buffer or for convenience use a buffer supplied by manufacturer that has been tested for its efficiency 2 Conventional cloning vectors and applications a Different cloning vectors used for different applications plasmids analyzing small DNA regions expressing genes in cell viruses cloning larger regions lambda virus gene therapy adenovirus artificial chromosome vectors BACs PACs YACs cloning chromosomal regions b Conventional E coli plasmid cloning vectors typically have origin of replication that functions in bacteria antibiotic resistance gene s selectable marker gene often lacZ encoding beta galactosidase polylinker also known as a multiple cloning site example pUC18 or pUC19 One would typically clone gene into polylinker within marker gene using restriction enzymes and DNA ligase transform E coli competent cells then use antibiotic resistance and the marker phenotype to identify recombinants c Bacteriophage lambda is used to clone larger DNAs or complementary DNAs capacity about 10 20 kb replace internal section required for lysogeny but not for lytic life cycle with foreign DNA 2 d Cosmids capacity 30 44 kb plasmid vector with lambda packaging site cos not as popular for use because they are unstable and prone to rearrangements e M13 virus small single stranded virus of E coli replication intermediate RF is double stranded and can be used as cloning vector much like a plasmid after transforming E coli single stranded virus is produced single stranded DNAs readily sequenced using dideoxysequencing also provide a template for site directed mutagenesis protocols 3 Cloning vectors for genomics a Capacity of conventional cloning vectors limiting for studying genomes particularly the human genome b Large capacity vectors for cloning in bacterial make use of bacterial artificial chromosomes BACs and bacteriophage P1 artificial chromosomes PACs Figure of BAC 3 BAC Based on E coli fertility factor F factor Low copy number per cell 1 2 compared to conventional plasmid vectors F plasmid vectors can accept large fragments 300 kb Electroporation is used to introduce recombinants into E coli Produce low yields of recombinant DNA PAC Based on E coli bacteriophage P1 Has a large genome that can accommodate 100 kb of DNA Recombinant P1 phage can be adsorbed onto E coli host New hybrid vectors have features of BACs and PACs combined c Yeast artificial chromosomes Can accommodate megabase 1000 2000 kb fragments Contain the critical elements of a yeast chromosome including a centromere two telomeres and autonomously replicating sequences ARS important for replication Yeast spheroplasts yeast with cell walls removed can take up YACs Yield of transformants and yield of DNA both low Especially important for mapping large genomic regions Figure of YAC 4