Biotechnology  Tools

Electrophoresis similar to SDS PAGE for proteins: (acrylamide--chemical crosslinks) usually on a horizontal agarose gel--melts at high temperature; solid at room temp detect DNA using ethidium bromide/UV light also detect using radioactivity (32P); fluorescence; chemiluminescence	nucleic acids are negatively charged, so move  toward + electrode according to their charge proportional to MW--separate according to size smaller NA move more quickly through gel than larger NA
Restriction enzyme--molecular scissors endonucleases--does not require an end (exonucleases) >100 restriction enzymes known names come from organism: EcoRI--E. coli  recognize a specific palindromic DNA sequence and cut the DNA palindrome is the same forwards/backwards some leave 3' overhang; 5' overhang or blunt ends overhangs leave--"sticky ends"--even though DNA is cut, can have base-pairing move DNA from one organism to another - "recombinant DNA" put DNA together with DNA ligase use synthetic DNA of desired sequence to "paste" on restriction site if nature did not provide one	methylation protects DNA from restriction enzymes mechanism for bacteria to protect itself from invading phage or other bacterial DNA
Plamids are cloning vectors plasmids are closed circular DNA, with origin of replication--replicated within bacteria to many copies carries a resistance gene--ampicillin, tetracyclin, kanamycin  take DNA from one organism, cut with RE, isolate fragment desired from a gel cut a plasmid or phage DNA with same RE put these two DNA fragments together via sticky ends, ligate them closed we have recombinant DNA this is transferred into bacterial cells by electroporation or chemical competence plate on media with antibiotic to kill bacteria that did not take up a plasmid--no proof that your foreign DNA is there, only that the plasmid is there individual colonies contain a single plasmid
How do you know your foreign DNA was inserted? one method:  interrupt a gene that is a reporter -  b-galactosidase (lacZ) use a substrate for b-galactosidase that when cleaved give a colored compound do this on antibiotic media to select for plasmid induce the gene with a lactose-analog if the gene is intact get blue color--no foreign insert, just plasmid if the gene has an insert (foreign DNA) then the reading frame is thrown off and no b-galactosidase is produced--no color
phage may be used as vectors phage are bacterial viruses accept larger pieces of DNA same procedures of cut/paste package phage DNA into virus particles infect a lawn of bacteria phage infection lyses bacteria, clear area "plaque" each plaque represents a single phage
DNA Libraries--what is the purpose of  the library? genomic DNA that has been fragmented by RE will include introns from eukaryotes                     cDNA made from mRNA for eukaryotes solve the problem of introns similar methods of cut/paste into desired vector:  plasmid vs. phage phage easier to screen larger numbers; larger inserts; pain to isolate and purify DNA from plasmids are easier to work with once identified
Screening of colonies/plaques how do you find the gene you want? need a probe a similar gene from another organism if the genes are similar enough synthetic DNA based upon a protein sequence need to take into account the degeneracy of genetic code label the probe with radioactivity or a molecule that can be detected (e.g. biotin) transfer plaques or phage to a solid support (nitrocelluose or nylon) denature DNA into single strands probe will anneal to DNA it is complimentary to detect which plaque or colony contains desired DNA retrieve the plaque or colony from original plate
Applications:  What can you do with a gene once you have it? ask bacteria to make the protein for you design in the appropriate promoter, Shine-Delgarno sequences to get transcription/translation insulin is made by bacteria make chains separately; join by disulfide bonds human growth hormone; tissue plasminogen activator/enterokinase (dissolve blood clots); erythropoietin (stimulates RBC production); many others
Transgenic Organisms put the gene back into an organism put the gene into the germ line; inject DNA into the nucleus of fertilized eggs; recombination search out the gene in the population of offspring this gene is now carried in the germ line and passed to succeeding generations use similar techniques to knockout a gene and ask what effect it has on the organism	Agricultural Transgenic Plants take a gene from one organism and put in a different type artic fish anti-freeze protein and put into frost-sensitive plants --protects from freezing insect toxin, Bt, express in plant to prevent insect damage:  corn, cotton insert resistance to herbicides--soybeans improve nutritional quality--"golden" rice--vitamin A in rice prevent viral diseases learn more about agricultural biotechnology
Is this bad, are these Frankenfoods? plants and animals have been bred for thousands of years for desirable traits trial and error to observe/produce the trait wanted biotech simply shortens the time needed to introduce the trait
Compare enegy needed to grow potatoes conventionally with pesticides vs with Bt do we need to be cautious--yes--products should be well tested before released there are many benefits to be gained better nutrition fewer chemical pesticides pharmaceuticals--edible vaccines be informed and make a rational choice
PCR--polymerase chain reaction** easy way to amplify specific regions of DNA
DNA sequencing** determine the precise order of the bases uses DNA polymerase, DNA primer (synthetic oligonucleotide of defined sequence), radioactive or fluorescently labeled dXTPs low levels of dideoxy nucleotides to randomly stop elongation of DNA--once dideoxy is incorporated, no OH for chain elongation represent every possible position separate on a gel to see relative positions and read by "ladder" sequencing method
Gene Mapping where is a gene on a chromosome bacterial genes were mapped on how long it took for the gene to be transferred into another bacteria during conjugation bacteria can exchange genetic information problem with antibiotic resistance and toxin genes compare the genetic map wit the physical map made from restriction fragments
Mapping of eukaryotic chromosomes more difficult chromosome walking--end to end matching of DNA fragments RFLPs--2 chromosomes--DNA in may vary in RE site from one chromosome to the other lose a site or gain a site due to mutation in a gene different lengths of "filler" DNA distinguish individuals by cutting DNA with RE and comparing patterns--forensics this can be done with genomic DNA and detect with a probe  use PCR to amplify a specific region--compare sizes of product