Module 1.2 Basic Principles of DNA Recombinant Technology Flashcards

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1
Q

Characteristics of a recombinant DNA molecule

A
  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
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2
Q

Characteristics of rDNA technology:

A
  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
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3
Q

Recombinant DNA technology is made possible by what enzymes?

A

Restriction endonuclease and ligases

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4
Q

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

A

restriction endonucleases

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5
Q

Explain the following terms related to restriction endonucleases:

Recognition sequence

Restriction site

A

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

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5
Q

Which class/es of restriction endonuclease require ATP?

A

Types I

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6
Q

Which class of restriction endonuclease is most specific? Least specific

A

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

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7
Q

Give examples of restriction endonucleases

A

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

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

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8
Q

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

A

ligase

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8
Q

What are the non-specific counterparts of Restriction Endonucleases?

A

Deoxyribonucleases and Ribonucleases

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9
Q

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

A

Stanley N. Cohen

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10
Q

What is the first plasmid bacterial cloning vector isolated?

A

λdvgal 120 (isolated from bacteriophage lambda)

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11
Q

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

A

galactose metabolism

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12
Q

λdvgal 120 was concatenated with what genetic material?

A

Simian Vacuolar Virus 40 (SV40) genome

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13
Q

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

A

plasmid-Stanley Cohen 101 (pSC101),

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14
Q

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

A

severe combined immunodeficiency disorder

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15
Q

Differentiate reproductive cloning and molecular cloning

A

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

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

16
Q

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

A

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.

17
Q

The process of recombinant DNA technology:

A
  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.
18
Q

Helps synthesize DNA strands

A

polymerase

18
Q

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

A

restriction enzymes

19
Q

Two types of restriction enzymes

A

endonuclease - within sequence

exonuclease - from ends

20
Q

Components of the a plasmid vector

A

origin of replication
selectable marker
cloning sites

20
Q

Two common vectors for rDNA technology

A

plasmid
bacteriophages

20
Q

Helps bind two pieces of DNA into one

A

ligases

21
Q

Ways in which rDNAs are inserted into the host include

A

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

21
Q

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

A

vector

22
Q

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

A

cloning sites

23
Q

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

A

Host organism

24
Q

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

A

microinjection

25
Q

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

A

gene gun

26
Q

Explain how the use of Ca2+

A

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

26
Q

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.

A

Heat shock; alternate cooling and heating

26
Q

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

A

electrophoration

27
Q

What is the applications of recombinant DNA technology in pharmacy?

A

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
28
Q

Application of recombinant DNA technology in gene therapy:

A
  1. Insertion of tumor suppressor genes
  2. Oncolytic virotherapy
  3. Gene-directed enzyme prodrug therapy
29
Q

Application of recombinant DNA technology in clinical diagnosis:

A
  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
30
Q

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

A

tomato CGN-89564-2

31
Q

Several challenges associated with rDNA technology

A
  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
32
Q

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.

A

delayed softening