BCH 313 Recombinant DNA

Question 1

Definition of recombinant DNA

Answer 1

According to the NIH guidelines, recombinant DNA are molecules constructed outside of living cells by joining natural or synthetic DNA segments to DNA molecules that can replicate in a living cell or molecules that result from their replication

Recombinant DNA, also known as in vitro recombination, is a technique involved in creating and purifying desired genes.

Question 2

Overview

Answer 2

Isolate DNA

Cut with restriction enzymes

Ligate into cloning vector

Transform recombinant DNA molecule into host cell

Each transformed cell will divide many, many times to form a colony
of millions of cells, each of which carries the
recombinant DNA molecule :

Question 3

Summary of the process of making recombinant DNA

Answer 3

1. Treat the DNA taken from both sources with the same restriction endonuclease.
2. The restriction enzyme cuts both molecules at the same site.
3. The ends of the cut have an overhanging piece of single-stranded DNA called “sticky ends.”
4. These sticky ends are able to base pair with any DNA molecule that contains the complementary sticky end.
5. Complementary sticky ends can pair with each other when mixed.
6. DNA ligase is used to covalently link the two strands into a molecule of recombinant DNA.
7. To be useful, the recombinant DNA needs to be replicated many times (i.e. cloned). Cloning can be done in vitro, via the Polymerase Chain Reaction (PCR), or in vivo (inside the cell)
   using unicellular prokaryotes (e.g. E. coli), unicellular eukaryotes (e.g. yeast), or mammalian tissue culture cells.

Question 4

Some of the molecular biology techniques utilized during recombinant DNA include

Answer 4

1. The study and/or modification of gene expression patterns.
2. Gene cloning
3. DNA sequencing
4. Creation of transgenic plants and animals

Question 5

The study and/or modification of gene expression patterns.

Answer 5

Gene expression is the process by which a gene’s coded information is converted into the structures present and operating in the cell. Expressed genes include those that are transcribed into mRNA (messenger RNA) and then translated into protein, and those that are transcribed into tRNA (transfer RNA) and rRNA (ribosomal RNA). Gene expression can be studied using microarray analysis, which is a method of visualizing the patterns of gene expression of thousands of genes using fluorescence or radioactive hybridization.

Question 6

Gene cloning

Answer 6

Gene cloning utilizing recombinant DNA technology is the process of manipulating DNA to produce multiple copies of a single gene or segment of DNA.

Question 6

Example of use of Recombinant DNA Technology

Answer 6

Insulin Production

The DNA for insulin is first isolated

A plasmid made of DNA is removed from a bacterial cell

A restriction enzyme cuts the plasmid DNA open, leaving sticky ends.

The insulin gene, with complementary sticky ends, is added.
DNA ligase enzyme splices (joins) together the plasmid DNA and the insulin DNA.

The plasmid (now genetically modified) is inserted back into the bacterium.

The bacterium host cell, divides and produces copies of the plasmid.

The Bacterium makes human insulin using the gene in the plasmid.

The insulin is extracted from the bacterial culture.

Question 6

Creation of transgenic plants and animals

Answer 6

A transgenic plant or animal is one that has been genetically engineered, and usually contains genetic material from at least one unrelated organism, such as from a virus, other plant, or other animal.

Question 6

DNA sequencing

Answer 6

DNA sequencing is a lab technique used to determine the sequence of nucleotide bases in a molecule of DNA.

Question 7

Creation of transgenic plants and animals

Answer 7

A transgenic plant or animal has been genetically engineered and usually contains genetic material from at least one unrelated organism, such as from a virus, other plant, or other animal.

Question 8

Example of use of Recombinant DNA Technology/ medical application

Answer 8

Insulin Production
1. The DNA for insulin is first isolated
2. A plasmid made of DNA is removed from a bacterial cell
3. A restriction enzyme cuts the plasmid DNA open, leaving sticky ends.
4. The insulin gene, with complementary sticky ends is added.
5. DNA ligase enzyme splices (joins) together the plasmid DNA and the insulin DNA.
6. The plasmid (now genetically modified) is inserted back into the bacterium.
7. The bacterium host cell, divides and produces copies of the plasmid.
8. The Bacterium makes human insulin using the gene in the plasmid.
9. The insulin is extracted from the bacterial culture.

Question 9

Gene therapy

Answer 9

Gene therapy is the introduction of normal genes into individuals who have defective genes.

Question 10

Genetic expression

Answer 10

This involves converting code on a gene to structures that can be observed, this can be seen in the conversion of DNA to mRNA.

Question 11

Vector

Answer 11

A carrier DNA molecule to which the fragment of DNA of interest is attached

Question 12

5 medical applications of recombinant DNA

Answer 12

Molecular basis of the disease
Diagnosis of disease
Production of proteins
Gene therapy
Transgenic animals
Genetic counseling
in forensic medicine
commercial applications

Question 13

Molecular basis of disease

Answer 13

Recombinant DNA
technology is used to understand the molecular basis of a number of diseases, for example:
– Familial hypercholesterolemia
– Sickle cell disease
– Thalassemias
– Cystic fibrosis
– Muscular dystrophy, etc

Question 14

Production of proteins

Answer 14

Using recombinant technology, human proteins can be produced in abundance for therapy, research and diagnosis, for example:
– Anticoagulant: Tissue plasminogen activator
(TPA). Used in treating heart attack victims.
– Human growth hormone: It is used to treat
children with growth hormone deficiencies
(dwarfism)
– Erythropoietin: Used to treat anemia.
– Blood factors: Factor VIII, used to treat
hemophilic patients.
– Insulin: A hormone used to treat diabetes.
– Interferons: Used to treat cancer.
– Interleukins: Used in wound healing, HIV
infections, cancer, immune deficiencies.
– Monoclonal antibodies: Used in diagnostic tests.
– Superoxide dismutase: Used during surgery.
– Vaccines: Various vaccincs can be produced by
recombinant DNA technology. The first
successful recombinant DNA vaccine produced
was for the hepatitis B virus.

Question 15

Gene therapy

Answer 15

Gene therapy is the introduction of
normal genes into individuals who have defective genes. Gene therapy for sickle cell disease, thalassemia, adenosine deaminase deficiency and other diseases may be devised. Currently, gene therapy is at the experimental level.

Question 16

Transgenic animals

Answer 16

Genes can be introduced into fertilized eggs from which transgenic animals develop and these transgenic animals can produce normal offspring. Transgenic means containing
genetic material into which DNA from a different the organism has been artificially added.

Question 17

Genetic counseling

Answer 17

Genetic counseling is the device of preventing passing of defective genes to offspring.
Screening tests, based on the recombinant DNA techniques, can be performed on the prospective parents prior to conception. If they have decided to conceive, the fetus can be tested for genetic defect.
If the fetus has the defect, treatment can be started at an early stage, even in utero.

Question 18

In forensic medicine

Answer 18

Special techniques, e.g. DNA fingerprint is used in forensic medicine and can be used in criminal cases.

Question 19

Commercial applications

Answer 19

– Agricultural application: In agriculture, plants that are resistant to disease, insects, herbicides, drought and temperature extremes or more efficient at fixing nitrogen are being produced using recombinant DNA technology.

– Industrial applications: Industrial applications includes the production of enzymes used in detergents, sugar and cheese.