Transcript Molecular Techniques in Cell & Molecular Biology

Molecular Techniques in Cell
& Molecular Biology
BSI 420
Lecture 6
Sept. 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