3 22 2014 CHAPTER 20 Analyzing and Engineering Genes genetic engineering manipulation of DNA sequences in organisms o recombinant DNA technology techniques used to engineer genes ability to create novel combinations of DNA sequences by cutting specific sequences and pasting them into new locations recombinant DNA strategy for producing human growth hormone involved cloning the human gene introducing gene into bacteria and having recombinant bacterial cell produce the hormone GH1 codes for pituitary growth hormone when not present pituitary dwarfism occurs o o o o o o o reverse transcriptase enzyme that synthesizes DNA from RNA template o researchers used enzyme to make complementary DNA cDNA from mRNA isolated from pituitary cells cDNA is any DNA made from an RNA template DNA cloning process of producing many identical copies of a gene used by researches to copy cDNAs for analysis and determine which encoded growth hormone protein plasmids small circular DNA molecules that replicate independently of the chromosome often found in bacteria serve as a vector vehicle for transferring recombinant genes to a new host if a recombinant plasmid is inserted into bacterial yeast cell foreign DNA is copied then transmitted to new cell as host cell grows and divides used to produce millions of identical copies of specific genes restriction endonucleases bacterial enzymes that cut DNA at recognition sites specific base sequences recognition sites are palindromes often produces sticky ends complementary single stranded ends that are produced when enzymes make staggered cuts in the DNA if restriction sites in different DNA sequences are cut w same restriction endonuclease makes the same sticky ends in both samples allows resulting fragments to be spliced together by complimentary base pairing cloning genes into plasmids involves cutting the plasmid and cDNA with same restriction endonuclease sticky ends of plasmids and cDNAS bind by complementary base pairing DNA ligase seals recombinant pieces of DNA together by forming phosphodiester bond linkage transformation process of taking up DNA from the environment and incorporating it into the genome DNA library a collection of transformed bacterial cells each containing a vector w an inserted gene important bc give researchers a way to store information from a particular cell type or genome in accessible form genomic library made up of cloned DNA fragments represents an entire genome o may be made by isolation of mRNA synthesizing cDNA making cDNA double standed making recombinant plasmid transformation cDNA library collection of bacterial cells each containing a vector w one cDNA o researchers inferred approximate sequence for GH1 gene from amino acid sequence of human growth hormone made a probe based on inferred sequence and radioactively labeled it cDNA library screened by a probe in search of the plasmid containing GH1 cDNa once human growth hormone cDNA isolated cloned into plasmid under control of bacterial promoter transformed E coli cells produced human growth hormone that could be isolated and purified DNA probe single stranded fragments of a known gene that binds a complementary sequence in the single stranded DNA being analyzed o o o o o o o o 3 22 2014 o must be labeled so it can be found after it has bound the target sequence ethical concerns over recombinant growth hormone FDA approved use only for children projected to reach adult heights of 5 3 for males and 4 11 for females polymerase chain reaction PCR in vitro DNA synthesis reaction where a specific DNA sequence is replicated repeatedly generates many identical copies of a particular DNA sequence possible only when DNA sequence information surrounding the gene is available requires primers that match sequences on both sides of the gene they will bind to single stranded target DNA one primer is complementary to a sequence on one strand upstream of the target DNA other primer complementary to sequence on the other strand downstream of the target undergoes steps to occur steps 2 4 continually repeated to yield necessary of copies reaction mix prepared containing dNTPs a DNA template copies of the two primers and Taq mixture heated to 95oC two strands of DNA consequentially separated mixture cooled primers allowed to bond anneal to complementary sections of single stranded step 1 polymerase step 2 denaturation step 3 primer annealing target DNA step 4 extension mixture heated to 72oC causing Taq polymerase to synthesize complementary DNA strand from dNTPs beginning primer dideoxy sequencing a method for determining DNA sequence based on an in vitro DNA synthesis rxn developed by Sanger to understand more about gene function carried out by adding ddNTPs and dNTPs to synthesis rxns ddNTPs identical to dNTPs except they lack 3 hydroxyl group so DNA polymerization stops once a ddNTP is added to a growing strand current technique uses fluorescent markers for each ddNTP allows DNA to be sequenced w one dideoxy rxn instead of 4 genetic map linkage map meiotic map used to locate gene genes associated with a phenotype such as a disease contain genetic markers genes or other loci that have known locations single nucleotide polymorphisms SNPs sites in DNA where some individuals in population have different bases may be used as genetic markers each genetic marker provides a landmark at a position along a chromosome that is known relative to other markers must be polymorphic 2 different phenotypes exist in same population of species phenotype associated w marker must be variable DNA samples from affected families may be analyzed with genetic markers to follow inheritance of specific DNA regions genes closely linked if a certain marker and certain phenotype are almost always inherited together o genes involved are physically close to each other on same chromosome o to locate specific diseases one must find a large of affected and unaffected people genetic marker occurs in affected individuals but not unaffected people physical map currently used records absolute position of a gene along a chromosome in s of base pairs gene therapy introduction of a gene into affected cells to replace augment defective copies w normal alleles healthy allele must be sequenced an well understood o o DNA has to be introduced so that gene expression occurs in the correct issues amount time o retroviruses current choice of vector in gene therapy contain an RNA genome and the enzyme reverse transcriptase if human genes
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