GENETIC ENGINEERING
Chapter 14
Genetic engineering refers to the techniques used to modify the DNA of an organism to produce genes
with new characteristics.
Recombinant DNA technology is used in genetic engineering to isolate and obtain many copies of a
DNA segment in order to study it biochemically or to combine or introduce foreign genes into an
organism.
These techniques have led to technologies that are being used extensively in medicine, pharmaceuticals,
agriculture and others.
RECOMBINANT DNA
Recombinant DNA is formed when a DNA sequence from another organism is incorporated into a
carrier or vector molecule, which is then introduced into a cell. This is called transformation.
The cell with the vector will multiply the recombinant DNA during cell mitosis.
Restriction enzymes are used to cu DNA into specific fragments.
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Each restriction enzyme cuts DNA at a highly specific base sequence.
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Many restriction enzymes cleave DNA sequences to produce complementary single-stranded
cut ends called sticky ends or palindromic sequences.
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DNA ligase is the enzyme to recombine fragments.

Restriction enzymes can recognized anywhere from 4 to 23 bases sequences, called
restriction sites.
Restriction sites are scattered throughout the DNA strand.
Bacteriophages and plasmids are the most commonly used vectors.

Bacteriophages are viruses that attack and destroy bacteria.

Plasmids are circular DNA strands found in bacteria, different from the bacterial
chromosome.
The size of DNA fragments is measured in kilobases, 1000 bases = 1 kb.
CLONING OF DNA.
Genome is the total genetic material of an organism; the total DNA present in a cell.
A genomic library is a collection of DNA fragments that are more or less representative of the entire
DNA in the genome.
Each fragment is spliced into a plasmid, which is kept inside a bacterial cell.
Genes from eukaryotes contain introns and cannot be expressed properly. Bacterial cells lack the enzyme
needed to remove introns.
Splicing DNA into a vector.
1. The plasmid and the DNA from another organism are treated with the same restriction enzyme.
2. Single molecules with complementary single-stranded sticky ends are produced.
3. The two types of molecules are mixed together so that their sticky ends pair.
4. The mixture is then treated with DNA ligase so the phosphodiester bonds are formed at the
junctions. The new plasmid DNA is called recombinant DNA.
5. The plasmid are inserted into E. coli. Each bacterium accepts only one plasmid. Not all cells take
in a plasmid.
6. E. coli is then treated with an antibiotic that will kill those cells that did not accept the plasmid.
7. When E. coli divides and multiplies, it also reproduces the plasmid. In this way the recombinant
DNA is amplified or cloned.
A specific DNA sequence can be detected by a complementary genetic probe, a radioactive single
stranded DNA sequence.
Complementary DNA library (cDNA).
Reverse transcriptase can be use to make DNA copies of mRNA isolated from eukaryotic cells.
The resulting fragments is called a cDNA library. This complementary library is called so because it is
complementary to the mRNA.
These fragments are then incorporated into recombinant vectors.
Introns have been removed from the mRNA and the genes in these fragments can be expressed by E. coli.
Amplifying DNA in vitro.
The polymerase chain reaction is a technique used to amplify DNA in vitro.
1. The DNA double helix is separated by heating.
2. A chemically synthesized primer with a specific sequence is introduced and becomes attached to the
complementary DNA sequence.
3. Primer attachment designates the starting point of replication by DNA. polymerase.
4. The process is repeated several times, each time doubling the number of DNA molecules.
After 20 cycles of heating and cooling there are 220 copies of the target sequence.
The synthetic primer is prepared to recognize a target sequence, the sequence to be replicated.
The DNA polymerase used is heat resistant. It has been extracted from bacteria living in hot springs of
Yellowstone National Park.
Gel electrophoresis.
Gel electrophoresis is used to separate macromolecules.
Charged molecules can be separated based on the rate at which they migrate in an electrical field.
Nucleic acids are negatively charged and migrate to the positive pole.
The gel slows down larger molecules more than the small molecules.
The rate at which the molecules travel is inversely proportional to the length of the molecule.
Procedure:
1. An electric field is set up in a gel material consisting of agarose or polyacrylamide, which is poured
as a thin slab on a glass or Plexiglas holder.
2. After the gel has solidified, samples containing a mixture or macromolecules of different sizes are
loaded in wells formed at one end of the gel.
3. An electrical current is applied. The smallest fragments travel the longest distance.
4. The gel containing the separated DNA fragments is treated with ethidium bromide, a dye that binds to
DNA and is fluorescent under UV light.
In the Southern blot technique,
1. DNA fragments are separated by gel electrophoresis and then blotted onto a nitrocellulose filter.
2. A radioactive probe is then hybridized by complementary base pairing to the DNA bound to the
filter.
3. The radioactive bands can be identified by autoradiography.
Northern blot is used to study RNA fragments.
The Western blot is used to study polypeptides already separated by gel electrophoresis.
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The polypeptides of interest are identified by radioactive antibody molecules that bind to
them specifically.
DNA sequencing
The degree of genetic relationship among the individuals in a population can be determined by comparing
nucleotide sequences.
Procedure:
1. Dideoxynucleotides are modified nucleotides that lack a 3’ hydroxyl group and thus block further
elongation of a new DNA chain. Four different ones are used: ddATP, ddCTP, ddGTP and ddTTP
2. Four different reaction mixtures are prepared. Each mixture of containing single-stranded DNA
fragments, all deoxynucleotides needed for DNA synthesis, DNA polymerases, radioactive labeled
primers and only one of the dideoxynucleotides.
3. The dideoxynucleotide will be incorporated in any location where the DNA fragment has its
complementary base. A series of DNA fragments are created ending at all the possible positions
where the complementary base was found in the original DNA molecule.
4. The products are separated by gel electrophoresis and the position of the fragments is determined by
autoradiography.
Restriction fragments length polymorphism.
RFLP is also use to determined genetic relationship between individuals.
Procedure:
1. Restriction enzymes are used to cut DNA from two or more individuals.
2. The fragments are separated by gel electrophoresis with the DNA of each individual on a different
lane.
3. A Southern blot is made on the gel after hybridization with radioactive probes.
4. The resulting bands are identified and compared between individuals.
APPLICATIONS OF GENETIC ENGINEERING.
Recombinant DNA and genetic technology is used to devise new combinations of genes to produce
improved pharmaceutical and agricultural products.
Transgenic organisms have incorporated foreign DNA into their cells.
Genes isolated from one organism can be modified and expressed in other organisms ranging from
bacteria to transgenic plants and animals.
Expression of eukaryotic genes in bacteria requires that the gene be linked to regulatory elements that the
bacterium can recognize.
Bacterial cells do no have the enzymes needed for porttranscriptional and posttranslational processing of
eukaryotic proteins.
Expression of eukaryotic genes in eukaryotic hosts shows promise because the machinery for processing
proteins is already present.