Molecular Biology and W. H. Freeman
CHAPTER 14 LECTURE NOTES: RECOMBINANT DNA TECHNOLOGY
I.
General Info
A. Landmarks in modern genetics
1. Rediscovery of Mendel’s work
2. Chromosomal theory of inheritance
3. DNA as the genetic material
4. Recombinant DNA technology development and applications
B. Recombinant DNA refers to the creation of new combinations of DNA segments that are not found together in nature. The isolation and manipulation of genes allows for more precise genetic analysis as well as practical applications in medicine, agriculture, and industry.
C. Fundamental changes in our society are occurring as a result of genetic engineering.
II.
Making recombinant DNA
Overview: Isolate DNA à Cut with restriction enzymes à Ligate into cloning vector à transform recombinant DNA molecule into host cell à each transformed cell will divide many, many times to form a colony of millions of cells, each of which carries the recombinant DNA molecule (DNA clone)
(From: AN INTRODUCTION TO GENETIC ANALYSIS 6/E BY Griffiths, Miller, Suzuki,
Leontin, Gelbart © 1996 by W. H. Freeman and Company. Used with permission.)
A. Isolating DNA
1. Crude isolation of donor (foreign) DNA is accomplished by isolating cells à disrupting lipid membranes with detergents à destroying proteins with phenol or proteases à degrading RNAs with RNase à leaving DNA at the end
2. Crude isolation of plasmid vector DNA is accomplished by an alkaline lysis procedure or by boiling cells which removes bacterial chromosomal DNA from plasmid DNA.
3. To get purer DNA from either (1) or (2), crude DNA is
a) Fractionated on a CsCl2 gradient
b) Precipitated with ethanol
c) Poured over a resin column that specifically binds DNA
B. Cutting DNA
1. DNA can be cut into large fragments by mechanical shearing.
2. Restriction enzymes are the scissors of molecular genetics. Restriction enzymes (RE) are endonucleases that will recognize specific nucleotide sequences in the DNA and break the DNA chain at
I.
General Info
A. Landmarks in modern genetics
1. Rediscovery of Mendel’s work
2. Chromosomal theory of inheritance
3. DNA as the genetic material
4. Recombinant DNA technology development and applications
B. Recombinant DNA refers to the creation of new combinations of DNA segments that are not found together in nature. The isolation and manipulation of genes allows for more precise genetic analysis as well as practical applications in medicine, agriculture, and industry.
C. Fundamental changes in our society are occurring as a result of genetic engineering.
II.
Making recombinant DNA
Overview: Isolate DNA à Cut with restriction enzymes à Ligate into cloning vector à transform recombinant DNA molecule into host cell à each transformed cell will divide many, many times to form a colony of millions of cells, each of which carries the recombinant DNA molecule (DNA clone)
(From: AN INTRODUCTION TO GENETIC ANALYSIS 6/E BY Griffiths, Miller, Suzuki,
Leontin, Gelbart © 1996 by W. H. Freeman and Company. Used with permission.)
A. Isolating DNA
1. Crude isolation of donor (foreign) DNA is accomplished by isolating cells à disrupting lipid membranes with detergents à destroying proteins with phenol or proteases à degrading RNAs with RNase à leaving DNA at the end
2. Crude isolation of plasmid vector DNA is accomplished by an alkaline lysis procedure or by boiling cells which removes bacterial chromosomal DNA from plasmid DNA.
3. To get purer DNA from either (1) or (2), crude DNA is
a) Fractionated on a CsCl2 gradient
b) Precipitated with ethanol
c) Poured over a resin column that specifically binds DNA
B. Cutting DNA
1. DNA can be cut into large fragments by mechanical shearing.
2. Restriction enzymes are the scissors of molecular genetics. Restriction enzymes (RE) are endonucleases that will recognize specific nucleotide sequences in the DNA and break the DNA chain at