Summary
Summary of Recombinant DNA lecture series
- Course
- Biochemistry (MCB2021F)
- Institution
- University Of Cape Town (UCT)
Notes contain the whole section covered in Recombinant DNA.
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Recombinant DNA technology:A fragment of DNA that includes the gene to be cloned is inserted into a circular DNA
molecule called a vector, to produce a recombinant DNA molecule. The vector transports
the gene into a host cell, which is usually a bacterium, although other types of living cells
can be used.
Within the host cell the vector multiples, producing numerous identical copies, not only of
itself but also of the gene it carries. When the host cell divides, copies of the recombinant
DNA molecule are passed to the progeny and further vector replication occurs.
After a large number of cell divisions, a colony/ clone of identical host cells is produced.
Each cell in the clone contains one or more copies of the recombinant DNA molecule; the
gene carried by the recombinant molecule is now cloned.
Recombinant DNA technology within reach à disease diagnostics, forensics, law
Application of recombinant DNA technology:
Basic principles of recombinant DNA technology:
- Isolate DNA (copy, PCR)
- Cut the DNA at a known palindromic cleavage
site using restriction enzymes
- Use ligases to glue back together the DNA into
the new combinations (with inserted DNA)
- Introduce the recombined DNA into an organism
for the replication and reproduction to occur
- Select for the desired genotype/ phenotype
- There is a huge advance over prior technology
e.g. breeding/ crossing, random mutation and
selection.
,Construction of a recombinant DNA molecule à
Why recombinant DNA technology is important:
Usually only on recombinant DNA molecule is transported into any single
host cell, so that although the final set of clones may contain many different
recombinant DNA molecules, each individual clone contains multiple copies
of just one molecule.
The gene is now separated away from all the other genes in the original
mixture, and its specific features can be studied.
Tools needed in R-DNA technology:
DNA sequence, restriction enzyme (Res), Vectors (Plasmid, Bacteriophage, Cosmid, BACs),
DNA ligase, Reverse transcription, Host organism (alive), DNA polymerase and Alkaline
phosphatase.
DNA sequence:
DNA is the raw material needed, it is heated so that it can
be denatured. The DNA strands separate when heated to
just below boiling point, this exposes the nucleotides and
when slowly cooled, the strands renature due to
complementary base pairing.
Total cell DNA is DNA from a source of material of which the genes of interest are to be
cloned from, this can be DNA from a bacteria culture, plant, animal cell or any other
organism. It consists of genomic DNA from the organism along with any additional DNA
molecules such as plasmids.
, Pure vector DNA comes from a phage or plasmid, the DNA needs to prepared from a culture
of bacteria, which follows the same basic steps as purification of total cell DNA. The crucial
difference is that at some stage the plasmid DNA must be separated from the main bulk of
chromosomal DNA which is in the cell.
Preparation of cell extract:
E. coli bacteria that is used, the cell wall needs to be weakened by ethylenediamine
tetraacetate (EDTA), this removes the magnesium ions which are essential for preserving
the overall structure of the cell envelope, and also inhibits cellular enzymes that can
degrade DNA.
The detergent sodium dodecyl Sulphate (SDS) is used to aid cell lysis by removing lipid
molecules and thereby disrupting the cell membrane. It forms mixed micelles, molecules
that absorb lipids
Sodium hydroxide (NaOH) disrupts cell wall, alkaline pH loosens the wall and it also disrupts
hydrogen bonds that hold together DNA double helix.
Removal of protein by phenol extraction:
Concentration of DNA:
The most frequent used method of concentration is ethanol precipitation. DNA is
polar due to charged phosphate backbone. In the present of salt (mono-covalent
cations Na+) and at a temperature of <-20oC, absolute ethanol efficiently
precipitates polymeric nucleic acids.
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