FUNDAMENTALS OF BIOTECHNOLOGY

FUNDAMENTALS OF BIOTECHNOLOGY Lecture # 08 Study!! • Linkers & Adaptors • Selection and Characterization of rDNA • cDNA Libraries • DNA transformation Linkers and Adaptors • Sticky ends are desirable for DNA cloning experiments. • Provided by treating the target and vectors with same R.E or with different but producing the sticky end. • But some time target DNA blunt ended • So therefore we will have to use Linkers and Adaptors. Linkers • Synthetic , Short and known double stranded oligonucleotides sequence. • Having blunted ends on both sides and R. Sits. • Treatment with R.E produces sticky ends after ligation with target DNA. • e.g. Linker having sit for BamHI. • Drawback if target DNA also having the same R. Site then? • (Please use the book notes for detail). [...]... dideoxynucleotides as chain terminators to produce a ladder of molecules generated by polymerase extension of primer DNA libraries DNA libraries  DNA libraries are sets of DNA clones, each of which has been derived from the insertion of a different fragment into a vector followed by propagation in the host  A collection of DNA fragments representing the genome of an organism inserted in a suitable vector  A... vector or include elements required for expression • Select for restoration of lost function Southern and Northern blotting Identify the protein product of an interested gene Western blotting using a specific antibody PCR (polymerase chain reaction) to amplify a sequence of DNA using a pair of primers each complementary to one ends of the target DNA sequence Running on Gel separation size based Nucleic... Electrophoresis Hybridization to identify the interested DNA or its RNA product 1 Radiolabeled probes which is complementary to a region of the interested gene Probes:  An oligonucleotide derived from the sequence of a protein product of the gene  A DNA fragment/oligo from a related gene of another species 1 Blotting the DNA or RNA on a membrane 2 Hybridize the labeled probe with DNA membrane (Southern) or RNA... organism inserted in a suitable vector  A clone is a genetically distinct individual or set of identical individuals DNA libraries Genomic libraries : Genomic DNA library represents total genomic DNA prepared form random fragments of genomic DNA, which may be inefficient to find a gene because of the huge abundance of the non-coding DNA cDNA libraries : cDNA library represents expressed genes (reverse... transcription are inserted into a vector to form a cDNA library Large genomes need large numbers of clones to achieve high coverage Number of colonies needed for 95 % coverage: Species (35 kb) E coli S Cerevisiae D melanogaster Rice Human Frog Genome size (bp) 4.6 x 106 1.8 x 107 1.2 x 108 5.7 x 108 3.2 x 109 2.3 x 1010 number of clones 410 1500 10000 49000 274000 1969000 To reduce clone numbers in a library: increase...Screening and characterization of rClones • Next step is to know which one contains our DNA of interest • Genetic Method (Resistance, Color, plaque formation) • Immunological screening: (inserted gene will produce protein Ags and Abs reaction based) • Hybridization