What is a Mutation? Biotechnology, Tools





 What is a Mutation?

1) Mutations are changes in the genetic sequence, and they are a main cause of diversity among organisms. 

2) A single mutation can have a large effect, but in many cases, evolutionary change is based on the accumulation of many mutations with small effects. 


3) Mutations effects can be beneficial, harmful, or neutral depending on their context or location.

Example: Sixkle cell anemia disease in humans caused due to single gene mutation. 


Sickle cell anemia

1) It is an inherited blood disorder in affected individuals at birth, causing the production of abnormal haemoglobin.

2) Normally, the haemoglobin protein which resides insides red the lungs and carries it to all parts of the body.

3)  Healthy red blood cells are flexible so that they can move through the smallest blood vessels. 


4) In sickle cell disease, the haemoglobin is abnormal, causing the red blood cells to be rigid and shaped like a "C" or Sickle, the shape from which the disease takes its name.

5) Sickle cells can get stuck and block blood flow, causing pain and infections. 



Eugenics, Euthenics, and euphemism 

1) Eugenics: 

Eugenics is the practice or advocacy of improving the human species by selectively mating people with specific desirable hereditary traits. 

2) It aims to reduce human suffering by " breeding out" diseases, disabilities, and so _ called undesirable characteristics from the human population.

3 Euthenics:

Euthenics is a branch of science that aims to better different aspects of the environment to improve humans, wellbeing/ or the wellbeing of other living things. 

● Water treatment plants are examples of authenies in action.

4 The euphemism 

The euphemism deals with the control of several inherited human diseases, especially inborn errors of metabolism in which the missing or defective enzyme has been identified. 

BIOTECHNOLOGY 

Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans.

The two core techniques that enabled birth of modern biotechnology are:

1) Genetic Engineering  :

Techniques to alter the chemistry of genetic material ( DNA and RNA ) to introduce these into host organisms and thus change the phenotype of the host organism. 

2) Bioprocess Engineering: 

Maintenance of sterile ( microbial contamination _ free) ambience in chemical engineering processes to enable growth of only the desired microbe / Eukaryotic cell in large quantities for the manufacture of biotechnologies products like antibiotics, vaccines, enzymes, etc.

Recombinant DNA : A DNA molecule made is vitro with segment from different sources. 

TOOLS OF RECOMBINANT DNA TECHNOLOGY 

1.Restriction Enzymes: 

Restriction enzymes belong to a larger class of enzymes called nucleases.

● These are of two kinds; exinucleases and endonucleases.


■ Endonucleases remove nucleotides from the ends of the DNA whereas endonucleases make cuts at specific positions within the DNA.

■ Restriction endonucleases are used in genetic engineering to form ' recombinant ' molecules of DNA, which are composed of DNA from different sources/ genomes. 

2) Separating DNA molecules:

The fragments produced by restriction enzymes would not be of much use if we could not also easily separate them for analysis. 

● The most common separation technique used is gel electrophoresis. 

● This technique takes advantage of the negative charge on DNA molecules by using an electrical field to provide the force necessary to separate DNA molecules based on size.

● The separated DNA fragments can be visualized only ofter staining the DNA with a compound known as  ethidium bromide followed by exposure to UV radiation. 

What is a Plasmid? 

A Plasmid is a small circular piece of DNA found in bacteria cells that is physically separated from chromosomal DNA and can replicate independently. 



3) cloning vectors 

Vectors used at present,  are engineered in such a way that they help easy linking of foreign DNA and selection of recombinant from non _ recombinants.

Feature of a vector:


■ origin of replication ( ori): 

This is a sequence from where replication starts and any piece of DNA when linked to this sequence can be made to replicate within the host cells.

■ Selectable marker:

Which helps in identifying and eliminating non trans formants and selectively permitting growth of the transformations. 


■Transformation is a procedure through which a piece of DNA is introduced in a host bacterium. 

■ Cloning Sites: 

To link the alien DNA, the vector needs to have few, preferably single recognition sites for the commonly used restriction enzymes. 

Methods of insertion of DNA in Host cells:

1) Micro _ injection:

Recombinant DNA is directly injected into the nucleus of an animal cell.

 Balistics or gene gum:

Cells are bombarded with high velocity micro_ particles of gold or tungsten coated with DNA in a method known as biolistic or gene gum.

This method is suitable for plants.



Basic process in making recombinant DNA molecules:

Recombinant DNA technology involves several steps in specific sequence such as:


■ Isolation of the Genetic Material ( DNA).

■ Cutting of DNA at specific locations: Restriction enzyme digestion are performed by incubating purified DNA molecules with the restriction enzyme.

■.Amplification of Gene of interest using PCR:

PCR stands for polymerase Chain Reaction. 

■ PCR is a laboratory method used to make many copies of a specific piece of DNA from a sample that contains very tiny amounts of that DNA.

■ Insertion of Recombinant DNA into the Host cell / organisms: 

Insert a piece of alien DNA into a cloning vector and transfer it into a bacteria a plant animal cell, the alien DNA gets multiplied. 

■ Obtaining the foreign Gene produce:

The Transfer Cells produce products of genes of interest. This should be isolated for further uses.

Other Types of vectors 

1). Expression vector:

Expression vector which are created particularly to express the transgene in the target cell, typically have a promoter region that drives transgene expression. 



2). Transcription vectors:

Contrary to expression vector, simpler vectors known as transcription vectors are only able to replicate in a target cell but cannot express themselves. Their insert is amplified by means of transcription vectors.

3). Cosmid : 

A hybrid vector known as  a Cosmid contains the cos gene from the Lambda phage. They are frequently employed in genetic engineering as a cloning vector. 

4). Fosmid:

To enable the cloning of huge DNA segments, fosmids are DNA vectors that utilize the F_ Plasmid origin of replication and partitioning mechanisms. 

A variety of applications have led to the development and adaptation of vectors _ This are two main uses: 

■ To separate, locate and store piece of a bigger genome;

 ■ To specifically express Gene's that code for certain proteins. 


Genome Sequencing:

All living things ( bacteria plants, and mammals) have a distinct genetic code, or genome, made up of nucleotides based ( A, T, C and G) . An  organisms own DNA fingerprint, or pattern can be detected if you know the basis sequence. 


■ Sequencing is the process of establishing the order of basis:

An organisms genomes base order can.be determined in one step via a laboratory approach called whole genome Sequencing. 

Steps of DNA sequencing:

1). DNA shearing:

To begin researchers use molecular scissors to cut the DNA which is made up of millions of base ( As,Cs, Ts, and Gs,) into manageable chunks that can be ready by a Sequencing machine.

2). DNA bar coding: 

To determine whether fragment of sheared DNA belong to bacterium, researchers add tiny pieces of DNA tags,or bar codes .This is comparable to how a grocery store bar code identifies a product. 

3). DNA Sequencing: 

Several bacteria's bar _ coded DNA is mixed and put into a DNA sequences. Each bacteria sequence's A, C, T and G bases , by the sequence. To keep track of which based belong to which bacteria, the sequencer uses the bar _ code.

4). Data analysis:

To compare sequences from various bacteria and spot discrepancies, scientists employ computer analysis techniques. 



 
The DNA Technology ( use and application) Regulation Bill 2019, was recently submitted by the parliamentary Science and Technology committee. 

■ The Bill's goal is to govern how DNA information is used to determine people's identities.

The purpose of these profiles is to aid low enforcement agencies in their investigations. 

 What are the Salient features of the Bill?

Salient features of the Bill: 

1). Use of DNA Data: 

Under the Bill, DNA testing is allowed only in respect of matters listed in the schedule to the Bill.These include offences under the Indian penal code, 1860 and for civil matters such as paternity suits. Further the schedule includes DNA testing for matters related to Establishment of individual identity. 

2). Collection of DNA: 

While preparing a DNA profile, bodily substances of persons may be collected by the investigating authorities. Authorities are required to obtain consent for collection in certain situations. 
■ For arrest persons, authorities are required to obtain written consent if the offense Carrie's a punishment of up to seven years. 
■ If the offence Carrie's more than seven years of imprisonment or death, consent is not required. 
■ Futher, if the person is a victim, or relative of a missing person, or a minor or disabled person, the authorities are required to obtain the written consent of such victim, or relative,or parent or guardian of the minor or disabled person.

■ If consent is not given in these case, the authorities can approach a Magistrate who may order the taking of bodily substances of such persons. 

3) DNA Data Bank: 

The Bill Provides for the establishment of a Nation DNA Data Bank and Regional DNA Data Banks, for every state, or two or more states .DNA laboratories are required to share DNA data prepared by them with the National and Regional DNA Data Banks.

4). Removal of DNA profiles: 

The Bill states that the criteria for entry, retention, or removal of the DNA profile will be specified by regulations.
■ However,the Bill provides for removal of the DNA profiles of the following persons; (i) of a suspect if a police report is filed or court order given (ii) of  an under trial if a court order is given,and ( iii) on written request for persons who are not a suspect, offender or under trial.

5) DNA Regulatory Board:

The Bill provides for the establishment of a DNA Regulatory Board, which will supervise the DNA Data Banks and DNA laboratories. 

6) DNA laboratories: 

Any laboratory DNA testing is required to obtain accreditation from the Board.

■ The Board may revoke the accreditation  for reasons including failure to : (i) undertake DNA testing or (ii) comply with the conditions attached to the accreditation. 

7). Offences: 

The Bill specifies penalties for various offences, including: (i) for disclosure of DNA information, or (ii) using DNA samples without authorization. For instance, disclosure of DNA information will be punishable with imprisonment of up to three years and fine of up to one lakh rupees.



Issues associated with this Bill

1) : consent clause: 

The Magistrate can easily overturn it, making consent a formality .when the magistrate can overrule consent, there is  no advise on the grounds and reasons for doing so.

2)   Data Protection: 

Due to the lack of strong data protection regulations, there are worries about the security of many DNA profiles that will be stored at the National DNA Data Bank and its regional centres.

3) issues of Misuse: 

Allowing investigating agencies to collect DNA samples from " Supects", will give them " unbridled power that is easily capable of misuse and abuse " and amount to a " threat to the life, liberty, dignity and privacy of a person. 


4) creating separate data banks for civil and criminal matters. " This will result in a presumption against the person, even through they have not consented to giving their DNA sample for use in the criminal investigation. 

Way forward

1) Adequate provisions for cyber security 

To avoid cyber security breaches, DATA banks and laboratories should have the greatest levels of cyber security feasible. 

2) Strict Legislation for Data Protection:

India initially needs legislation to protect personal data and privacy, as well as sample collecting that is well _ defined and purpose _ driven 

3) Need for independent security:

Proposals to destroy Biological materials and erase DNA profiles from the database must be subjected to independent assessment. 

4) Make provisions for the removal of a person's DNA profile from the data bank as soon as he or she is declared innocent. 


RNA interference 

1) It is a gene _ silencing technique that uses doublestanded RNA to prevent protein production in target cells.

2) RNAi takes place in all Eukaryotic organisms as a method of cellular Defence. 

3) This method involves silencing of a specific mRNA due to a complementary Double standard RNA ( ds RNA) molecule that binds to and prevents translation of the mRNA ( silencing).

4) This natural mechanism for sequence specific gene silencing promises to revolutionize experimental biology and may have important practical applications in functional genomics, therapeutic intervention agricultura, and other areas. 

Production of pest resistant plants using RNAi

1) Several nematodes parasites a wide variety of plants and animals including human being.

2) A nematodes Meloidegyne incognita infects the roots of tobacco plants and causes a great reduction in yield.

3) A novel strategy was adopted to prevent this infection which was based on the process of RNA interference ( RNAi)

4) Using Agrobacterium vectors, nematode _ specific Gene's were introduced into the host plant.


5) The introduction of DNA was such that it produced both sense and antisense RNA in the host cells. This two RNA's being complementary to each other formed a double stranded ( dsRNA)that initiated RNAi and thus, silenced the specific mRNA of the nematode.


6) The consequence was that the parasite could not Survive in a transgenic host expressing specific interfering RNA .structure of cells































































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