BIOL 311 Human GeneticsRecombinant DNA Methods: Restriction Enzymes, Cloning VectorsReading: Chap. 5 pp. 129-133; 139-146Lecture Outline:1. Restriction enzymes2. Conventional cloning vectors and applications3. Cloning vectors for genomicsLecture:1. Restriction enzymesa. Discovered in bacteria and blue-green algae.b. Enzymes that recognize and cleave specific sites on DNA. Are endonucleases--cleavewithin 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), createfragments for end-labeling--used in restriction mapping, genomic maps, RFLP analysis.Examples:EcoRI Escherichia coli R factor 5'G AATT C3' 5' overhang3'C TTAA G5'PstI Providencia stuarti 5'C TGCA G3' 3' overhang3'G ACGT C5'AluI Arthrobacter luteus 5'AG CT3' Blunt end3'TC GA5'Naming: 1st letter is genus name, 2nd and 3rd letter is species name, numbered in order of discovery.Small scale restriction digests: 20 ul reaction2 ul of 10x restriction buffer (buffer, Mg2+, salts)1-10 ul of DNA sample (0.5-1 ug)1 ul (1-2 units) restriction enzyme1Each 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 applicationsa. Different cloning vectors used for different applications:plasmids--analyzing small DNA regions, expressing genes in cellviruses--cloning larger regions (lambda virus), gene therapy (adenovirus)artificial chromosome vectors (BACs, PACs, YACs)--cloning chromosomal regionsb. Conventional E. coli plasmid cloning vectors typically have:origin of replication that functions in bacteriaantibiotic resistance gene(s)selectable marker gene (often lacZ, encoding beta-galactosidase)polylinker (also known as a multiple cloning site)example: pUC18 or pUC19One would typically clone gene into polylinker within marker gene using restriction enzymes and DNA ligase, transform E. coli competent cells, then use antibiotic resistanceand the marker phenotype to identify recombinants.c. Bacteriophage lambda is used to clone larger DNAs or complementary DNAscapacity about 10-20 kbreplace internal section required for lysogeny, but not for lytic life cycle, with foreign DNA2d. Cosmidscapacity 30-44 kbplasmid vector with lambda packaging site (cos)not as popular for use because they are unstable and prone to rearrangementse. M13 virussmall single stranded virus of E. coli replication intermediate (RF) is double-stranded and can be used as cloning vector (muchlike a plasmid)after transforming E. coli, single stranded virus is producedsingle stranded DNAs readily sequenced using dideoxysequencing; also provide a template for site directed mutagenesis protocols.3. Cloning vectors for genomicsa. 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 3BAC: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 DNAPAC:Based on E. coli bacteriophage P1Has 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 chromosomesCan 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