Molecular Techniques in Cell & Molecular Biology BSI 420Lecture 6Sept. 19, 2002 “Insanity is Hereditary, You get it from your children” -Sam Levinson

RECOMBINANT DNA TECHNIQUES have revolutionized cell and molecular biology in the last 20 years. These techniques are used to recombine DNA from different sources and to replicate and express these genes in other cells. They make possible new ways to study the functions of genes and their protein products and also commercial production of specific gene products such as human insulin in bacteria. (We offer a full-semester course in RDNA techniques; this is by necessity a brief intro) 4 steps: 1. Recombination 2. Selection 3. Replication 4. Expression 1. Recombination Cut w Restriction Endonucleases – cut specific internal sequences Isolated from different bacteria and named after the source.

eg EcoR1 cuts to produce complementary or “sticky” ends. Because it recognizes and cuts this sequence of 6 (6 cutter), It makes med. -sized pieces. 4-cutter, small and 8 cutter, large pieces.

Cut DNA can be recombined w other DNA cut w the same endonuclease.

b.. Incorporation into vector DNA 1) Plasmid – a small circular DNA molecule thet can replicate in bacteria separately from the regular genome A plasmid cloning vector has an ORI to be able to replicate, and one or more antibiotic resistance genes that allow selection. Plasmids cut with one nuclease will hybridize with pieces of DNA cut with the same nuclease, due to the sequences being complementary. Then DNA ligase forms covalent phosphodiester bonds to create one DNA molecule from two.

2) Viruses like phage lambda or SV40 can also be used to clone DNA. 3) Yeast artificial chromosomes can be used to clone very large pieces of DNA

2. Selection a. Chemical selection – by antibiotic resistance E.g.Plasmid w ampicillin resistance allows cells to grow and divide b. Physical selection - Plate bacteria at low density, grow clones, and detect recombinant genes by colony hybridization.

Can be used to isolate one gene from a library of all genes from an organism, called a genomic library.

Making cDNA

PCR

DNA Microarray