Biology 5 Marks Questions

	Recombinant Proteins	Therapeutic uses
(a)	Insulin	Used for diabetes mellitus
(b)	OKT-3	Therapeutic antibody, used for reversal of transplantation rejection
(c)	DNase	Treatment of cystic fibrosis
(d)	Reo Pro	Prevention of blood clots
(e)	Blood clotting factor VIII	Treatment of haemophilia A
(f)	Blood clotting factor IX	Treatment of haemophiiia B
(g)	Tissue plasminogen activator	For acute myocardial infarction
(h)	Interferon alpha (INF alpha)	Used for hepatitis C
(i)	Interferon beta (INF beta)	Used for multiple sclerosis
(j)	Interferon gamma (INF gamma)	Used for granulomatous disease

1.  

• Should have ori/origin of replication,
• Has selectable marker, genes encoding for an antibiotic resistance/genes encoding for a - galactosidase,
• Has cloningsite/recognition site, for the restriction enzyme to recognise.

2. DNA is a hydrophilic molecule

Bacterial cell is made competent by treating with specific concentration of Ca++ ion/divalent ions, incubating the mon ice, heat shock for a short period and placing it back on ice again.

1.  

• Identifying the restriction endonuclease that recognises the palindromic nucleotide sequence of the desired gene.
• The restriction endonuclease inspects the DNA sequences - finds and recognises the site.
• Cuts each of the double helix at the specific point - a little away from the centre of the palindromic site - between the same two bases on the opposite strand
• Makes the over hanging stretch single stranded portion as a sticky end.

2.  

• By PCR/Polymerase Chain Reaction.
• Desired gene is synthesised in vitro.
• DNA is denatured.
• Annealed using two sets of primers.
• Thermostable Taq polymerase extends the primers using nucleotides (provided in the reaction and genomic DNAas template).
• Amplified fragments are ligated.

1. Amplification of gene of interest using PCR is required.
2. Denaturation, annealing and extension are the basic steps of PCR.
3. Kary Mull is developed PCR in 1985.
4. Honesty and sense of responsibility towards his duty.

Upstream processing: Biotechnological processes can be separated into upstream processes and downstream processes. The upstream process is defined as the entire process from DNA isolation and culture expansion of the cells until final product.
Downstream processing: After completion of the biosynthetic stage, the product has to be subjected through a series of processes before it Is ready for marketing as a finished product. The processes include separation and purification, which are collectively referred to as downstream processing. The product has to be formulated with suitable preservatives. Such formulation has to undergo through clinical trials as in case of drugs. Strict quality control testing for each product is also required. The downstream processing and quality control testing vary from product to product.

1. Plasmid.
2. Cosmid.
3. Bacteriophage lambda.
4. BAC.

1. Restriction enzymes are derived from a defence unit called restriction modification system which protects the microorganisms from harm.
2. They are called as molecular scissors because they cut DNA molecules at specified location indicated by presence of specific recognition sequence.
3. No, the strands cut from within are then joined by another enzyme called DNA ligase. This joining is either with self or with another different strand.
4. Varun is curious, has scientific temperament and asks quick and intelligent questions.

1. Palindromes.
2. Restriction endonucleases.
3. EcoRI.
4. Sticky/ overhanding strands.

1.  

1. Engineered vectors help easy linking of foreign DNA.
2. They facilitate selection of recombinants from non-recombinants.

2. Features of Cloning vectors:

1. Origin of Replication (Ori):

• This is the sequence of DNA from where replication starts.
• Any piece of alien/ foreign DNA linked to it is made to replicate within host cell; it also decides the copy number of the linked DNA.

2. Selectable marker: A marker is a gene, which helps in selecting the host cells, which are transformants/ recombinants from the non-recombinant ones, eg ampicillin and tetracycline resistant genes in E.coli
3. Cloning site: The vector should have a few, preferably one unique recognition site to link the foreign DNA, presence of a particular cloning/ recognition site enables the particular restriction enzyme to cut the vector DNA.

rDNA is the DNA formed by combining DNAs from two different organisms. In bacterial cell, rDNA can be transferred by using a vector where it multiplies into many copies. These copies are preserved as gene library.
Recombinant DNA technology involves the following steps:

1. Isolation of DNA.
2. Fragmentation of DNA by restriction endonucleases.
3. Isolation of a desired DNA fragment.
4. Amplification of the gene of interest.
5. Ligation of the DNA fragment into a vector.
6. Insertion of recombinant DNA into the host.
7. Culturing the host cells on a suitable medium at a large scale.
8. Extraction of the desired gene product.
9. Downstream processing of the products as finished product, ready for marketing.

1.  

• The purified DNA molecule containing the desired gene is incubated with the specific restriction endonuclease, at optimal conditions of pH, temperature, etc. of that enzyme.
• Similarly, the vector DNA is also incubated with the same restriction endonuclease under optimal conditions.
• This results into cutting of the DNA into fragments.

2.  

• Gel electrophoresis is used to separate the DNA fragments produced by restriction digestion.
• The DNA fragments resolve according to their sizes, through the sieving effect provided bythe agarose gel.

3. The resultant fragments are joined to the vector by use of DNA-ligases.

Genetic engineering is a manipulation of genetic material in vitro. It involves the techniques to alter the chemistry of genetic material (DNA and RNA), to introduce the host organisms and thereby change the phenotype of the host organism.
Steps of DNA technology are:

1. Isolation of DNA.
2. Fragmentation of DNA by restriction endonucleases.
3. Isolation of the desired DNA fragments.
4. Amplification of the gene of interest.
5. Ligation of the DNA fragment into the vector using DNA ligase.
6. Transfer of the recombinant DNA into the host.
7. Culturing the host cell on a suitable medium on a large scale.
8. Extraction of the desired product.
9. Downstream processing of the products as finished products.

This process was designed by K. Mullis.

Isolation of the genetic material (DNA):

• RNA is removed by treatment with ribonuclease and proteins are removed by treatment with protease.
• After several treatments, the purified DNA is precipitated by adding chilled ethanol.
• The bacterial/ plant/ animal cell is broken down by enzymes to release DNA, along with RNA, proteins, polysaccharides and lipids.
• Bacterial cell is treated with enzyme lysozyme.
• Plant cell is treated with enzyme cellulase.
• Fungal cell is treated with chitinase.

Cutting of DNA at specific locations:

• The DNA is cut using restriction enzymes.
• The purified DNA is incubated, with the specific restriction enzyme at conditions optimum for the enzyme to act. Isolation of desired DNA fragment
• Using agarose gel electrophoresis, the activity of the restriction enzymes can be checked.
• Since the DNA is negatively charged, it moves towards the positive electrode or anode and in the process, DNA fragments separate out based on their sizes.
• The desired DNA fragment is eluted out.

Amplification of gene of interest using PCR:

• The Polymerase Chain Reaction (PCR) is a reaction in which amplification of specific DNA sequences is carried out in vitro.

1. Host cells are incubated with rDNA on ice.
2. Followed by placing them briefly at 42°C.
3. Then transfer them back on ice.

This enables the host cells (bacteria) to take up the rDNA.

Methodology used:

• Sequence Annotation-total DNA from a cell is isolated, converted into random fragments of relatively smaller sizes, and cloned in suitable host using specialized vectors.
• The cloning resulted into amplification of each piece of DNA fragment.
• The fragments were sequenced using automated DNA sequencers, these sequences are then arranged based on some overlapping regions (present in them).
• This requires generation of overlapping fragments (for sequencing).
• Specialised computer based programmes were developed, and these sequences were subsequently annotated and assigned to each chromosome.

Selection of recombinants due to inactivation of antibiotics is a laborious process as it requires:

1. A vector with two antibiotic resistance markers.
2. Preparation of two kinds of media plates, with one antibiotic each.

Transformed cells are first plated on the antibiotic plate which has not been insertionally inactivated (say, ampicillin) and incubated overnight for growth of transformants. For selection of recombinants, these transformants are replica-plated on second antibiotic (say, tetracycline) plate (which got inactivated due to insertion of gene). Non-recombinants grow on both the plates (one carrying ampicillin and the other carrying tetracycline) while recombinants will grow only on ampicillin plate.
This entire exercise is laborious and takes more time (two overnight incubation) as well. However, if we choose insertional inactivation of a marker that produces colour in the presence of a chromogenic compound, we can distinguish between the recombinants and non-recombinants on a single medium plate (containing one antibiotic and the chromogenic compound) after overnight growth.

In this method a recombinant DNA is inserted in the gene which inactivates the functioning of gene. Insertional inactivation is a efficient method to identify transformants.
Example:
Blue-white selection method: In this method, cloned DNA is inserted into the lac Z gene present on the vector. After insertion the lac Z gene is inactivated. The lac Z gene forms β−galactosidase enzyme which can break X-gal (a synthetic colourless substrate) into a blue coloured product. Thus, transformants will contain interrupted lac Z gene which will not produce β−galactosidase, appear in white colonies in the presence of X-gal.

Bacterial cell is made competent so that it can take up DNA. Taking up DNA is not easy as it is hydrophilic molecule and cannot pass through cell membrane. Bacterial cell can be made competent by treating it with a specific concentration of a divalent cation such as calcium as it increases the efficiency with which DNA can enter the bacteria through it the pores of cell wall.

Most commercial cloning vectors have key features that have made their use in molecular biology so widespread.
Control of Expressions: In the case of expression vectors, the main purpose of these vehicles is the controlled expression of a particular gene inside a convenient host organism (e.g. E. coli). Control of expression can be very important; it is usually desirable to insert the target DNA into a site that is under the control of a particular promoter. Some commonly used promoters are T7 promoters, lac promoters and cauliflower mosaic virus's 35s promoter (for plant vectors).
Selectable Marker: To allow for convenient and favorable insertions, most cloning vectors have had nearly all their restriction sites engineered out of them and a synthetic multiple cloning site (MCS) inserted that contains many restriction sites. MCSs allow for insertions of DNA into the vector to be targeted and possibly directed in a chosen orientation. A selectable marker, such as an antibiotic resistance is often carried by the vector to allow the selection of positively transformed cells. All plasmids must carry a functional origin of replication (ori).

A marker gene helps in differentiating between transformant genes and non-transformant genes. This helps in selecting the suitable recombinants. In case of E.coli; pBR322 is the vector for resistance to antibiotic tetracycline. The insertional inactivation of pBR322 will result in loss of resistance to tetracycline by E.coli. This can be found out by growing the recombinants on two plates; one containing tetracycline and another containing ampicillin. The recombinant will grow in ampicillin but not in tetracycline. A marker gene for chromogenic substrate helps in identifying the recombinant DNA on the basis of gain or loss of colour from the chromogenic substrate. Insertional inactivation of a marker gene coding for a chromogenic substrate will result in no blue colour imparted in the colony.
These steps are taken for easy identification of recombinants from non-recombinants.

1. Restriction endonucleases.
2. Agarose gel.
3. DNA - ligase.
4. Plasmid.
5. Hind II.
6. Elution.

Small volume cultures cannot yield appreciable quantities of products. To produce in large quantities, the development of bioreactors, where large volumes (100-1000 litres) of culture can be processed, was required. Thus, bioreactors can be thought of as vessels in which raw materials are biologically converted into specific products, individual enzymes, etc., using microbial plant, animal or human cells. A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen).
Structure of Bioreactor:

• It is a cylindrical structure with a curved base.
• A stirrer is present for even mixing and oxygen availability throughout the reactor.
• There is an agitator system, an oxygen delivery system, a foam control system, a temperature control system, etc.
• There is a sampling port through which small volumes of culture can be taken out periodically.

A flask in a laboratory cannot be used for producing recombinant DNA on large scale. Unlike a bioreactor; a flask cannot be used to grow culture continuously.
Difference:

	Flask		Bioreactor
i.	Flask is used to small laboratory scale testing of a culture.	i.	Bioreactor is used for commercial scale production.
ii.	The cells harbouring cloned genes of interest may be grown on a small scale in the laboratory.	ii.	The cells can also be multiplied in a continuous culture system wherein the used medium is drained out from one side while fresh medium is added from the other to maintain the cells in their physiologically most active log/ exponential phase. This type of culturing method produces a larger biomass leading to higher yields of desired protein.
iii.	Small volume cultures cannot yield appreciable quantities of products.	iii.	To produce in large quantities, the development of bioreactors, where large volumes (100–1000 litres) of culture can be processed, was required.