Download Recombinant DNA Technology:

Molecular biology lecture
Dr. Oruba Kuttof Hussein
Recombinant DNA Technology:
Recombinant DNA technology or Genetic engineering enables
scientists to study and experiment with DNA.
It is the direct human
manipulation of an organism's genome using modern DNA technology. It
involves the introduction of foreign DNA or synthetic genes into the
organism of interest. Scientists are using powerful techniques that act at the
molecular level to engineer crop plants and diagnose and treat diseases, also
called genetic modification,
Cutting DNA with restriction enzymes
Bacteria make enzymes called restriction endonucleases, or more
commonly, restriction enzymes, that cut strands of DNA into smaller pieces
(see figure -1). Bacteria use restriction enzymes to fight off attacking
viruses, chopping up the viral DNA so that the virus can’t destroy the
bacterial cell. Scientists use restriction enzymes in the lab to cut DNA into
smaller pieces so that they can analyze and manipulate DNA more easily.
Each restriction enzyme recognizes and cuts DNA at a specific sequence
called a restriction site
Figure 1: Restriction enzymes cut DNA only if the exact recognition
sequence is present:
As a result, restriction enzymes usually recognize palindromic sequences:
The restriction enzyme called EcoR1 cuts DNA at the sequence
5'GAATTC3'. If you mix DNA and a restriction enzyme, the enzyme will
find all the restriction sites it recognizes and cut the DNA at those locations.
Restriction enzymes make cutting and combining pieces of DNA easy. For
example, if you wanted to put a human gene into a bacterial plasmid, you’d
follow these steps:
1. Choose a restriction enzymes that forms sticky ends when it cuts DNA.
Sticky ends are pieces of single-stranded DNA that are complementary and
can form hydrogen bonds. Restriction enzymes that form sticky ends cut the
DNA backbone asymmetrically so that a piece of single stranded DNA
hangs off each end..
2. Cut the human DNA and bacterial plasmids with the restriction enzyme. If
you cut a plasmid DNA and human DNA with the same restriction enzyme,
all the DNA fragments will have the same sticky ends.
Some restriction enzymes, for example, Sma I, cut both DNA strands in the
middle of the recognition sequence and produce "blunt-end" DNA
fragments:
Cloning vectors
Formation of recombinant DNA requires a cloning vector, a DNA molecule
that will replicate within a living cell. Vectors are generally derived from
plasmids or viruses, and represent relatively small segments of DNA that
contain necessary genetic signals for replication, as well as additional
elements for convenience in inserting foreign DNA, identifying cells that
contain recombinant DNA, and, where appropriate, expressing the foreign
DNA. The choice of vector for molecular cloning depends on the choice of
host organism, the size of the DNA to be cloned, and whether and how the
foreign DNA is to be expressed.
In standard cloning protocols, the cloning of any DNA fragment essentially
involves seven steps: (1) Choice of host organism and cloning vector, (2)
Preparation of vector DNA, (3) Preparation of DNA to be cloned, (4)
Creation of recombinant DNA, (5) Introduction of recombinant DNA into
the host organism, (6) Selection of organisms containing recombinant DNA,
(7) Screening for clones with desired DNA inserts and biological properties.[
Selection
Not all the organism's cells will be transformed with the new genetic
material; in most cases a selectable marker is used to differentiate
transformed from untransformed cells. If a cell has been successfully
transformed with the DNA it will also contain the marker gene. By growing
the cells in the presence of an antibiotic or chemical that selects or marks the
cells expressing that gene it is possible to separate the transgenic events
from the non-transgenic. Another method of screening involves using a
DNA probe that will only stick to the inserted gene. A number of strategies
have been developed that can remove the selectable marker from the mature
transgenic plant