Module 1.2 Basic Principles of DNA Recombinant Technology

Question 1

Characteristics of a recombinant DNA molecule

Answer 1

1. Formed by laboratory methods of genetic recombination and molecular cloning
2. Genetic material from multiple sources
3. Creates a DNA molecule that would otherwise not be found in nature

Question 2

Characteristics of rDNA technology:

Answer 2

1. Alteration of genetic material outside an organism and introduction of the altered DNA fragment into organisms via an appropriate vector
2. Goal is to obtain enhanced and desired characteristics in living organisms or as their product
3. Method of joining two or more DNA molecules o create a hybrid
4. the result is a DNA molecule that does not exist in nature

Question 3

Recombinant DNA technology is made possible by what enzymes?

Answer 3

Restriction endonuclease and ligases

Question 4

These are enzymes recognizes a specific sequence of DNA and cuts, within, or close to that sequence

Answer 4

restriction endonucleases

Question 5

Explain the following terms related to restriction endonucleases:

Recognition sequence

Restriction site

Answer 5

Recognition sequence: sequence recognized by restriction endonuclease

Restriction site: location along the DNA where the cut is actually made, may within or outside the recognition site

Question 5

Which class/es of restriction endonuclease require ATP?

Answer 5

Types I

Question 6

Which class of restriction endonuclease is most specific? Least specific

Answer 6

Type II (25 bp resolution); Type I (1, 000 bp resolution)

Question 7

Give examples of restriction endonucleases

Answer 7

EcoR1 (G|AATTC) - from - E. coli R-strain

HindIII (A|AGCTT) - from - Haemophilus influenzae D-strain 3

Question 8

Joins together fragments of newly synthesized DNA to form a seamless strand

Answer 8

ligase

Question 8

What are the non-specific counterparts of Restriction Endonucleases?

Answer 8

Deoxyribonucleases and Ribonucleases

Question 9

This scientist was awarded the 1986 Nobel Prize for medicine in his discoveries on growth factors including nerve growth factors, and epidermal growth factors.

Answer 9

Stanley N. Cohen

Question 10

What is the first plasmid bacterial cloning vector isolated?

Answer 10

λdvgal 120 (isolated from bacteriophage lambda)

Question 11

The first cloning vector λdvgal 120 contains genes for what cellular process?

Answer 11

galactose metabolism

Question 12

λdvgal 120 was concatenated with what genetic material?

Answer 12

Simian Vacuolar Virus 40 (SV40) genome

Question 13

This new cloning vector was used to create intra- and interspecies rDNA from Salmonella.

Answer 13

plasmid-Stanley Cohen 101 (pSC101),

Question 14

The first approved gene transfer study was on a child with what disease?

Answer 14

severe combined immunodeficiency disorder

Question 15

Differentiate reproductive cloning and molecular cloning

Answer 15

Molecular cloning - creation of multiple copies of a fragment of DNA

reproductive cloning - creation of an exact copy of the whole multicellular organism

Question 16

Do not confuse recombinant DNA technology, molecular cloning, genetic engineering

Answer 16

recombinant DNA technology - a group of molecular techniques used to create recombinant DNA molecules by piecing together DNA fragments from multiple sources. The result is a chimeric DNA that is otherwise not found in nature

Molecular cloning - is a molecular technique used to create multiple copies of DNA molecules with desirable sequences. Often, these DNA molecules are in the form of vectors containing a gene of interest. This vector is introduced in a cloning host, usually a bacterium or yeast, allowing production of the exact copy of the DNA.

Genetic engineering is the process of using recombinant DNA technology to modify an organism’s DNA to achieve desirable traits. Addition of foreign DNA in the form of recombinant DNA vectors that are generated by molecular cloning is the most common method of genetic engineering.

Question 17

The process of recombinant DNA technology:

Answer 17

1. Isolate DNA containing gene of interest; isolate vector
2. Restriction digestion. Cut donor DNA and vector with the same restriction endonuclease.
3. PCR. After cutting, the donor DNA and the vector DNA can now be theoretically mixed to form a recombinant plasmid. However, some recombinant technology workstreams incorporate PCR to amplify the gene of interest after being subjected to restriction digestion such that a high number of the DNA of interest is produced. This ultimately increases the efficiency of the ligation process.
4. Ligation. In this step, the vector and the donor DNA are mixed to create a vector-donor hybrid by base-pairing of their sticky ends. The DNA hybrid is then covalently linked by DNA ligase that seals the nick in the sugar-phosphate backbone.
5. Insertion into cloning host. In this step, the recombinant plasmid is introduced into a cloning host (often bacterium such as E coli.) in a process known as transformation. The competency of the cloning host is one important factor to be optimized in this process, as incompetent cloning are less likely to take in the recombinant plasmid. To aid in the transformation process, techniques such as electrophoration, heat shock, or addition of Calcium phosphate are usually employed to increase the efficiency of transformation.
6. Isolation of transform cell. The result of transformation process is a mixture of transformed and untransformed cell. To isolate only the transformed cell, the cells are plated on a medium added with a particular antibiotic. The recombinant plasmid carried with it a selectable marker in the form a resistance genes against the antibiotic. As a result, only transformed cells that are expressing the recombinant plasmid can survive in the medium.
7. Expansion of the transformed cells. In this step, surviving colonies of transformed cells are expanded in large batch cultures. In the process, the recombinant plasmid is cloned in large amounts as the cells divides.
8. Isolation and cleaning of recombinant plasmid from expanded culture.
9. Transfer of recombinant plasmid in desired expression host. In this step, the extracted and cleaned recombinant plasmid is transferred to desired hosts to be expressed. The biological properties of the inserted gene is then tested. This may involve observing the phenotype of the cell or by extracting the expressed protein and subjecting it to some kind of functional assay.

Question 18

Helps synthesize DNA strands

Answer 18

polymerase

Question 18

Plays a major role in determining the location of a desired gene

Answer 18

restriction enzymes

Question 19

Two types of restriction enzymes

Answer 19

endonuclease - within sequence

exonuclease - from ends

Question 20

Components of the a plasmid vector

Answer 20

origin of replication
selectable marker
cloning sites

Question 20

Two common vectors for rDNA technology

Answer 20

plasmid
bacteriophages

Question 20

Helps bind two pieces of DNA into one

Answer 20

ligases

Question 21

Ways in which rDNAs are inserted into the host include

Answer 21

Microinjection
Biolistic gene gun
Heat shock (alternate cooling and heating)
Use of Ca2+ ions
Electroporation

Question 21

The ultimate vehicles that carry forward the desired gene into the host organism; helps in carrying and integrating the desired gene

Answer 21

vector

Question 22

sites recognized by the Restriction Enzymes wherein the desired DNAs are inserted

Answer 22

cloning sites

Question 23

Is the ultimate tool of rDNA technology wherein it takes in the vector engineered with the desired DNA with the help of enzymes

Answer 23

Host organism

Question 24

Involves the use of a glass needle to inject a genetic material into the Target Cell

Answer 24

microinjection

Question 25

Utilizes He gas to fire exogenous DNA strands onto the target Host Microorganism; usually done in plant cells

Answer 25

gene gun

Question 26

Explain how the use of Ca2+

Answer 26

Ca2+ coordinates DNA (negatively charged) and cell membrane (negatively charged) promoting intake of DNA either via diffusion or endocytosis.

Question 26

This involves subjecting host cells alternately in cold and hot temperature; such processes can lead to the formation of perforations to which genetic materials can enter into.

Answer 26

Heat shock; alternate cooling and heating

Question 26

Involves the use of electric fields to induce perforations within animal, bacteria or plant host cells

Answer 26

electrophoration

Question 27

What is the applications of recombinant DNA technology in pharmacy?

Answer 27

production of vaccines and protein therapies including:

1. human insulin
2. human growth hormone - for patients with pituitary gland disorders
3. Blood clotting factor VIII - protein replacement therapy for hemophilia patients
4. human follicle-stimulating hormone - treatment of infertility
5. Hep B virus surface antigens - for production of vaccines

Question 28

Application of recombinant DNA technology in gene therapy:

Answer 28

1. Insertion of tumor suppressor genes
2. Oncolytic virotherapy
3. Gene-directed enzyme prodrug therapy

Question 29

Application of recombinant DNA technology in clinical diagnosis:

Answer 29

1. Immunological assay commonly used to measure antibodies, antigens, proteins, and glycoproteins in biological samples
2. Diagnosis of HIV
3. Pregnancy tests
4. measurement of cytokines or soluble receptors in cell supernatant or serum

Question 30

Genetically modified ___ was the first commercially grown, genetically engineered crop product to be granted a license for human consumption

Answer 30

tomato CGN-89564-2

Question 31

Several challenges associated with rDNA technology

Answer 31

1. Post-Translational Modifications - cell stress response activation, instability and proteolytic activities, low solubility and resistance in expressing new genes in host cells
2. Mutations occurring in humans at the genetic level leading to deficiencies in protein production (in the context of gene therapy)
3. A Disaster for Safety and Biodiversity - possibility of genetically engineered plants cross-breeding with wild organisms
4. Dangerous Health Implications - can make deadly pathogens (i.e. Yersinia pestis) resistant to modern antibiotics and can be fatal to humans

Question 32

Developed in 1994, the genetically modified tomato was made to express the trait of ___ of tomato flesh as a practical means to minimize post-harvest crop losses.

Answer 32

delayed softening