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Genetic Engineering: Techniques and Impact

Molecular biology

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23 views4 pages

Genetic Engineering: Techniques and Impact

Molecular biology

Uploaded by

paulmorrisjohn
Copyright
© All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd

Genetic Engineering and future impact on life

Making recombinant DNA

Isolate DNA 

Cut with restriction enzymes. Crude isolation of donor (foreign) DNA is accomplished by
isolating cells , disrupting lipid membranes with detergents destroying proteins with phenol or
Proteases, degrading RNAs with RNase. Leaving DNA at the end.
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. For pure DNA,
crude DNA is a) Fractionated on a CsCl2 gradient, b) Precipitated with ethanol c) Poured over a
resin column that specifically binds DNA
5’--GAATTC--3’ 5’—G AATTC--3’
3’—CTTAA 3’—CTTAA G--5’

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 those points. A variety of
RE have been isolated and are commercially available. Most cut at specific palindromic sites in
the DNA (sequence that is the same on both antiparallel DNA strands). These cuts can be a
staggered which generate “sticky or overhanging ends” or a blunt which generate flush ends.
Joining DNA
Once you have isolated and cut the donor and vector DNAs, they must be joined together.
The DNAs are mixed together in a tube. If both have been cut with the same RE, the
ends will match up because they are sticky. DNA ligase is the glue of molecular genetics
that holds the ends of the DNAs together. DNA ligase creates a phosophodiester bond
between two DNA ends.
Amplifying the recombinant DNA

To recover large amounts of the recombinant DNA molecule, it must be amplified. This
is accomplished by transforming the recombinant DNA into a bacterial host strain.

The cells are treated with CaCl2


DNA is added
Cells are heat shocked at 42 C
DNA goes into cell by a somewhat unknown mechanism.
Once in a cell, the recombinant DNA will be replicated. When the cell divides, the replicated
recombinant molecules go to both daughter cells which themselves will divide later. Thus, the
DNA is amplified

Explanatory definitions

DNA clone = A section of DNA that has been inserted into a vector molecule and then
replicated in a host cell to form many copies.
Vectors
Requirements for a cloning vector
a) Should be capable of replicating in host cell
b) Should have convenient RE sites for inserting DNA of interest
c) Should have a selectable marker to indicate which host cells received
recombinant DNA molecule
d) Should be small and easy to isolate
Bacterial plasmids
They are small, circular DNA molecules that are separate from the rest of the chromosome. They
replicate independently of the bacterial chromosome. Useful for cloning DNA inserts less that 20
kb (kilobase pairs). Inserts larger than 20 kb are lost easily in the bacterial cell.

Future impact of Genetic Engineering

Therapeutic products Diagnosis Energy applications Genetically modified


(GM) products
Vaccines Gene therapy Biohydrogen Fruits
Growth hormones CRISPR Bioethanol GM vegetables
Recombinant protein Monitoring devices Biomethanol GM crops
Antibodies Therapeutic Biobutanol GM microbes
Vectors strategies GM animals
Anticancer drugs
Our DNA is made of four key molecules called bases (A, T, C and G). Sequences of these four
bases are grouped into genes. These genes act as the “code” for key substances the body should
make, such as proteins. Proteins are important molecules, vital for maintaining a healthy and
functional human being. Genes can be short, typically made of less than a hundred bases. A
good example includes ribosomal genes, which code for different ribosomes, molecules
which help create new proteins.

Long genes are made up of millions of bases. For example, the DMD gene codes for a protein
called dystrophin, which supports the structure and strength of muscle cells. DMD has over 2.2
million bases. Gene editing is a technology that can change DNA sequences at one or more
points in the strand. Scientists can remove or change a single base or insert a new gene
altogether. Gene editing can literally rewrite DNA.

The benefits of gene editing to humanity could be significant. For example, making a single base
change in people’s DNA could be a future treatment for sickle cell disease, a genetic blood
disease. People with this disease have just one base that has mutated (from A to T). This makes
the gene easier to edit compared with more complex genetic conditions such as heart disease or
schizophrenia.

Common questions

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Genetic engineering plays a vital role in energy applications by producing biofuels like biohydrogen, bioethanol, and biobutanol through engineered microbial pathways. These biofuels represent renewable energy sources that can reduce fossil fuel dependency, but their economic viability and environmental impacts require careful evaluation .

Genetic engineering facilitates the development of genetically modified organisms (GMOs), which offer benefits like improved crop yields and nutritional quality. However, ethical concerns include the impact on biodiversity, ecological balance, and long-term health effects. Balancing these benefits with ethical considerations necessitates careful regulation and public discourse .

Vectors in gene therapy serve as carriers that deliver edited genetic material into patient cells, crucial for treating genetic diseases by replacing or repairing defective genes. Their efficiency, specificity, and safety are critical parameters affecting the success and applicability of gene therapy interventions .

Recombinant DNA technologies facilitate the creation of therapeutic products like growth hormones, vaccines, and recombinant proteins by allowing precise manipulation and production of desired genetic sequences. These applications advance medical treatments by providing effective and scalable solutions .

A cloning vector must possess the ability to replicate within a host cell, convenient sites for restriction enzymes for DNA insertion, a selectable marker to identify host cells containing the recombinant DNA, and be small and easy to isolate. These features ensure that the inserted DNA can be efficiently replicated and identified in the host organism .

Restriction enzymes function as molecular scissors in genetic engineering by recognizing specific nucleotide sequences in DNA, typically palindromic sites, and cutting the DNA at these points. They can produce either staggered cuts which generate 'sticky' or overhanging ends, or blunt cuts that generate flush ends .

Unlike single-gene disorders, complex diseases like heart disease and schizophrenia involve multiple genes and environmental factors, presenting challenges for gene editing. Precision, off-target effects, and comprehensive understanding of these diseases complicate treatment. Advances in genomic research and targeted editing techniques are essential for addressing these complexities .

Amplification of recombinant DNA in a bacterial host involves transforming the DNA into bacteria treated with calcium chloride, followed by heat shock to facilitate DNA uptake. Once inside the cell, the recombinant DNA replicates along with cell division, passing replicated molecules to daughter cells and thus amplifying the DNA .

Gene editing can change DNA sequences, such as altering a single base, which is significant for diseases like sickle cell disease caused by a single nucleotide mutation (A to T). Correcting this mutation could potentially treat the disease, highlighting the precision and potential of gene editing for specific genetic disorders .

Bacterial plasmids are effective tools in cloning DNA segments less than 20 kilobase pairs due to their independent replication and ease of manipulation. However, they pose a limitation for larger inserts as these are often lost during replication in bacterial cells, likely due to instability or difficulty in replication management .

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