Genetic Engineering how to isolate large, pure quantities of a gene? could grow individual bacterial cells into large colonies could isolate plasmids from those cells could introduce plasmids into bacterial cells (transformation) could select for cells carrying a plasmid (resistance factors) needed a way to insert DNA into a plasmid Restriction enzymes, first discovered by Linn and Arber in the late '60s studied host restriction - some phage could only infect certain strains of bacteria bacterial strains had endonucleases that cut the viral DNA the bacterial chromosome was protected by methylation Hamilton Smith (1970) found the first restriction enzyme that cut a specific sequence in Haemophilus influenzae - HindII usually recognize palindromes type I cut a certain distance away from the palindrome type II cut in the palindrome may make a staggered cut, creates a sticky end Cohen and Boyer (1973) used EcoRI to combine two plasmids, the first in vitro produced recombinant DNA could select for the recombinants because one plasmid (pSC101) was resistant to tetracycline while the other (RSF1010) was resistant to streptomycin Try and answer this question about creating recombinant DNA from the Biology Project Cloning vectors Vectors for insertion of foreign DNA need an origin of replication, a selection mechanism and a cloning site many different ones with special properties plasmids such as pBR322, pUC19 small (as little as 3 kb) so transform well easy to isolate current ones have multiple cloning sites (MCS) disadvantage - because of transformation technique they can hold only small inserts (<10 kb) phage l can replace the lysogenic genes (in the middle of the phage) with up to 20 kb of DNA (must be 52 - 38 kb between cos sites) package the recombinant phage in vitro and use infection (much better than transformation) used for library construction m13 - a filamentous phage doesn't lyse the cell replicative form is double stranded but phage are single stranded - good for sequencing phagemid (pBluescript) - a combination of plasmid and f1 (phage) origin of replication cosmids - use cos ends and plasmid sequences to clone 40-50 kb of DNA YAC - yeast centromere, ARS (autonomous replication sequence), and telomere - can clone Mb size fragments in yeast expression vectors - have proper regulatory sequences so that introduced genes are expressed and translated shuttle vectors - can be moved between two different hosts (bacteria and yeast, for instance) How to clone a gene? Easy - make a library genomic (lambda or cosmid) shear or partially digest genomic DNA ligate to lambda or cosmid vector transform bacteria and select for recombinants advantages cloned the whole genome so the clone of interest must be in there gene regulatory regions will be included genomic DNA is easy to isolate disadvantages cloned the whole genome so have 100,000s of different clones to sort through clones include introns so can't be expressed in bacteria gene will be broken into pieces if it is very large cDNA (lambda or phagemid) isolate mRNA anneal oligo dT primer to poly A tail to provide a primer use reverse transcriptase to make a DNA copy of the RNA remove the mRNA with NaOH or RNAse use DNA polymerase I to synthesize the second strand the reverse transcriptase leaves a hairpin loop that can be used as a primer Use S1 nuclease to remove the hairpin loop ligate on linkers with restriction sites cut and ligate to vector (lambda or phagemid) transform bacteria and select for recombinants advantages includes only a small subset of the genes, only need to search through 10,000s of clones no introns, so clones can be expressed in bacteria much smaller inserts, so easier to grow and study disadvantages includes only a small subset of genes, your gene might not be in there no regulatory regions or introns so you really don't have the whole gene hard to isolate intact mRNA, especially if your gene is large Only one small problem, how to find the clone with the gene? Both of the above techniques produce a mixture of bacteria, each of which has a different cloned fragment inserted. Unfortunately, they are all mixed together and one bacterium looks pretty much like another (even to their mother). selection (not applicable for most eukaryotic genes) an assay (can be used to find genes for enzymes) use a probe RNA if you can purify the RNA DNA if it has been cloned in another species degenerate oligonucleotides if the protein has been sequenced antibodies and an expression library if antibodies specific for the protein product of the gene are available use the polymerase chain reaction to produce a probe chromosome walking and sequencing, if all you have is a phenotype (method of last resort) - cloning of Cystic Fibrosis gene is an example And the latest approach is to clone and sequence the whole genome, leading to the field of Genomics [ Biol 207 ] [ Bell ] [ CSU Chico ] [ Library ] This document is maintained by: Jeff Bell Last Update: Thursday, April 13, 2000