BIOTECH 5

 Amplification of Gene of Interest using PCR

• PCR stands for Polymerase Chain Reaction.
• Multiple copies of the gene of interest is synthesized in vitro using two sets of primers and the enzyme DNA polymerase.
• Primers are small chemically synthesized oligonucleotides that are complementary to the regions of DNA.
• PCR includes three major steps-

1. Denaturation

2. Annealing

3. Extension

• Denaturation is the process of heating of target DNA at 94oC to seperate the two strands of DNA.
• Annealing is the process of pairing of primers with complimentary base sequences of the two separated strands.
• Extension is the process of adding complimentary deoxyribonucleotides one by one to the 3/ OH ends of primers by the activity of DNA polymerase and as a result new DNA strand is synthesized.
• If the process of replication of DNA is repeated many times, the segment of DNA can be amplified to approximately billion times by the use of a thermostable DNA polymerase isolated from a bacterium, Thermus aquaticus.
• The amplified fragment can be used to ligate with a vector for further cloning.

Insertion of Recombinant DNA into the Host Cell/Organism

• Recipient cells after making them ‘competent’ to receive, take up DNA present in its surrounding.
• If a recombinant DNA bearing gene for resistance to an antibiotic is transferred into E. coli cells, the host cells become transformed into ampicillin-resistant cell.

Obtaining the Foreign Gene Product

• The foreign gene when gets expressed under appropriate conditions, produces desirable proteins.
• If any protein encoding gene is expressed in a heterologous host, is called a recombinant protein.
• The cells harboring cloned genes of interest may be grown on a small scale in the laboratory or on a large scale in a bioreactor.

Downstream Processing

• Downstream processing is the separation and purification of the product.
• The product has to be formulated with suitable preservatives and the formulation has to undergo thorough clinical trials.

BIOREACTORS

• Bioreactor is the cylindrical vessel in which biological processes is carried out on a large scale.
• The recombinant cells can 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.
• Bioreactors 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 such as temperature, pH, substrate, salts, vitamins, oxygen.
• Bioreactors are of two types-

1. Simple stirred tank bioreactor

2. Sparged stirred-tank bioreactor

• A stirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents and the stirrer facilitates even mixing and oxygen availability throughout the bioreactor.
• In sparged stirred-tank bioreactor sterile air is sparged through the reactor.
• The bioreactor has an agitator system, an oxygen delivery system and a foam control system, a temperature control system, pH control system and sampling ports so that small volumes of the culture can be withdrawn periodically.

BIOTECH 4

 

COMPETENT HOST

• Since DNA is a hydrophilic molecule, it cannot pass through cell membranes so the bacterial cells must first be made ‘competent’ to take up DNA.
• Several methods are followed to make the bacterial cells competent-
• Treating them with a specific concentration of a divalent cation, such as calcium, which increases the efficiency with which DNA enters the bacterium through pores in its cell wall.
• Recombinant DNA can then be forced into such cells by incubating the cells with recombinant DNA on ice, followed by placing them briefly at 42oC (heat shock), and then putting them back on ice which enables the bacteria to take up the recombinant DNA.
• Recombinant DNA can be directly injected into the nucleus of an animal cell by a method called micro-injection.
• In biolistics or gene gun method, cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA
• Disarmed pathogen vectors can be allowed to infect the cell to transfer the recombinant DNA into the host

PROCESS OF RECOMBINANT DNA TECHNOLOGY

Isolation of the Genetic Material (DNA)-

• The cells are broken and opened to release DNA along with other macromolecules such as RNA, proteins, polysaccharides and also lipids which can be achieved by treating the cells with enzymes such as lysozyme (bacteria), cellulase (plant cells), chitinase (fungus).
• The RNA can be removed by treatment with ribonuclease whereas proteins can be removed by treatment with protease and purified DNA ultimately precipitates out after the addition of chilled ethanol which can be seen as collection of fine threads in the suspension.

Cutting of DNA at Specific Location-

• Restriction enzyme digestions are performed by incubating purified DNA molecules with the restriction enzyme, at the optimal conditions for that specific enzyme which results in the fragments of DNA.
• The fragments are separated by a technique known as gel electrophoresis.
• Since DNA fragments are negatively charged molecules they can be separated by forcing them to move towards the anode under an electric field through agarose.
• The DNA fragments separate according to their size through sieving effect provided by the agarose gel.
• The smaller the fragment size, the farther it moves and the separated DNA fragments can be visualized only after staining the DNA with a compound known as ethidium bromide followed by exposure to UV radiation.
• Bright orange coloured bands of DNA can be observed in an ethidium bromide stained gel exposed to UV light.
• The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece by the process known as

BIOTECH 3

 

CLONING VECTORS

• A cloning vector is a small piece of DNA, taken from any organism into which a foreign DNA fragment can be inserted for cloning purposes.
• Plasmids and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomal DNA.
• If an alien piece of DNA with bacteriophage or plasmid DNA, we can multiply its numbers equal to the copy number of the plasmid or bacteriophage.

The following are the features that are required to facilitate cloning into a vector are-

• Origin of replication (ori)
• Selectable marker
• Cloning sites

Origin of replication (ori)- This is the 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-

• It helps in identifying and eliminating non transformants and selectively permitting the growth of the transformants.
• Transformation is a procedure through which a piece of DNA is introduced in a host bacterium.
• The genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc., are useful selectable markers for E. coli as the normal E. coli cells do not carry resistance against any of these antibiotics.
• Antibiotic resistance genes help in selecting recombinants from non-recombinants by a method called insertional inactivation where a recombinant DNA is inserted within the coding sequence of an enzyme β-galactosidase in the presence of a chromogenic substrate which results into inactivation of the enzyme.
• The presence of a chromogenic substrate gives blue colored colonies if the plasmid in the bacteria does not have an insert.
• Presence of insert results into insertional inactivation of the β-galactosidase and the colonies do not produce any colour, these are identified as recombinant colonies.

Cloning sites-

• Cloning sites are the recognition sites of the restriction enzymes.
• The ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes.

For example- ligation of a foreign DNA at the Bam H I site of tetracycline resistance gene in the vector pBR322

• Vector for cloning genes in plants is Agrobacterioum tumifaciens, a pathogen of several dicot plants which delivers a piece of DNA known as ‘T-DNA’ to transform normal plant cells into a tumor and direct these tumor cells to produce the chemicals required by the pathogen.
• The tumor inducing (Ti) plasmid of Agrobacterium tumifaciens has now been modified into a cloning vector.

• Vector for cloning genes in animals is retrovirus which transforms normal cells into cancerous cells.
• Retroviruses have been disarmed and used to deliver desirable genes into animal cells

BIOTECH 2

 

RESTRICTION ENZYMES

• Restriction enzymes belong to a larger class of enzymes called
• Restriction enzymes are called as molecular scissors because these enzymes cut DNA at specific sites.
• The first restriction endonuclease is Hind II.
• The restriction enzymes cut DNA at specific base sequence, and these specific base sequence is known as the recognition sequence.
• The convention for naming restriction enzymes –
• The first letter of the name comes from the genus.
• The second two letters come from the species of the prokaryotic cell from which they were isolated, e.g., EcoRI comes from Escherichia coli RY 13.
• In EcoRI, the letter ‘R’ is derived from the name of strain.
• Roman numbers following the names indicate the order in which the enzymes were isolated from that strain of bacteria.
• 900 restriction enzymes that have been isolated from over 230 strains of bacteria.

These are of two kinds

1. Exonucleases

2. Endonucleases

• Exonucleases remove nucleotides from the ends of the DNA whereas, endonucleases make cuts at specific positions within the DNA.
• Each restriction endonuclease recognizes a specific palindromic nucleotide sequences in the DNA.
• The palindrome in DNA is a sequence of base pairs that reads same on the two strands when orientation of reading is kept the same.

Example- the following sequences reads the same on the two strands in 5' à 3' direction, this is also true if read in the 3' à 5' direction.

 5' —— GAATTC —— 3’

 3' —— CTTAAG —— 5'

• Restriction enzymes cut the strand of DNA a little away from the center of the palindrome sites, but between the same two bases on the opposite strands which leaves a single stranded portions at the ends and the overhanging stretches called sticky ends on each strand.
• When cut by the same restriction enzyme, the resultant DNA fragments have the same kind of ‘sticky-ends’ and, these can be joined together using DNA ligases.

BIOTECH 1

 

INTRODUCTION

• Biotechnology deals with the techniques of using living organisms or enzymes from organisms to produce products useful to humans.

• The processes like in vitro fertilization leading to a ‘test-tube’ baby, synthesizing a gene and using it, developing a DNA vaccine or correcting a defective gene, are all parts of biotechnology.

• Biotechnology can be defined as- ‘The integration of natural science and organisms, cells, parts there of, and molecular analogues for products and services’.

PRINCIPLES OF BIOTECHNOLOGY

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

1. Genetic engineering

2. Maintenance of sterile conditions.

 

• Genetic engineering is the technique of altering the chemistry of DNA and RNA so that it can be introduced into the host organism to change the phenotype of the host organism.
• Sterile conditions should be maintained to enable growth of only the desired microbe or eukaryotic cell in large quantities for the manufacture antibiotics, vaccines, enzymes, etc.

Why we need genetic engineering??

• Hybridization (sexual reproduction)  procedures often lead to inclusion and multiplication of undesirable genes along with the desired genes.

• The techniques of genetic engineering which include creation of recombinant DNA, use of gene cloning and gene transfer allows us to isolate and introduce only one or a set of desirable genes without introducing undesirable genes into the target organism.

Problem - an isolated piece of DNA (containing desired gene) would not be able to reproduce itself in the progeny cells..

Solution - when it is integrated in the genome of recipient cells- multiply along with host DNA.

Three basic steps in genetically modifying an organism —

• Identification of DNA with desirable genes
• Introduction of the identified DNA into the host
• Maintenance of introduced DNA in the host and transfer of the DNA to its progeny.

TOOLS OF RECOMBINANT DNA TECHNOLOGY

Important tools of recombinant DNA technology are-

• Restriction enzymes- Restriction enzymes are called as molecular scissors because these enzymes cut DNA at specific sites.
• Cloning vector- Plasmids and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomal DNA.
• Competent host- The host should be competent enough to take up the foreign DNA.
• Bioreactors- Bioreactor is the cylindrical vessel in which biological processes is carried out on a large scale.

MOLECULAR BIOLOGY 14

 DNA fingerprinting

• The process of comparison of DNA from different sources to establish the identity is called DNA fingerprinting.

All persons 99.9% DNA same

0.1 % different and unique to individual - this is used in DNA fingerprinting.

Whole DNA sequence for differentiating 2 DNA would be very hectic.

• DNA fingerprinting involves identifying differences in some specific regions in DNA sequence called as repetitive DNA.

• Repetitive DNA are separated from bulk genomic DNA as different peaks during density gradient centrifugation.

• The bulk DNA forms a major peak and the other small peaks are referred to as satellite DNA.
• Means - satellite DNA contains - repetitive DNA

• Satellite DNA is of two types based on base composition, length of segment, and number of repetitive units
• micro-satellites
• mini-satellites

Satellite DNA

• Satellite DNA sequences normally do not code for any proteins, but they form a large portion of human genome.

• Satellite DNA sequence show high degree of polymorphism and form the basis of DNA fingerprinting.

• An inheritable mutation occurring in a population at high frequency, (more than 0.01- 1%) is referred to as DNA polymorphism.

• Repeated nucleotide sequences in the non-coding DNA of an individual is called Variable Number of Tandem Repeats (VNTR).

• The size of VNTR varies in size from 0.1 to 20 kb. (Kilo base pairs)

• DNA fingerprinting includes the following steps
• isolation of DNA
• digestion of DNA by restriction endonucleases
• separation of DNA fragments by electrophoresis
• transferring (blotting) of separated DNA fragments to synthetic membranes, such as nitrocellulose or nylon.
• hybridization using labelled VNTR probe
• detection of hybridized DNA fragments by autoradiography.

Fragments of DNA

Applications

• In identification of criminals.
• In determining population and genetic diversities.
• In solving parental disputes

MOLECULAR GENETICS 13

 Human genome project

• The scientific project which deal with the study of base sequences of DNA molecules of complete set of chromosomes is called human genome project.
• DNA -decides all property, so curiosity of knowing whole sequence.
• Genetic engineering techniques- DNA piece - isolate - sequencing. (Simple & fast)
• Launched in 1990

Initial problems-

1. 3×10`9 base pairs × initial estimated cost 3$ per base pair. = 9 billion $

2. Storage of info- 1000 latters in one page, 1000 pages per notebook, then it will require 3300 books..

• HGP was closely associated with the rapid development of a new area in biology- Bioinformatics.
• Goals of Human Genome project
• Identify all the approximately 20,000-25,000 genes in human DNA.
• Determine the sequences of the 3 billion chemical base pairs that make up human DNA.
• Store this information in databases;
• Improve tools for data analysis;
• Transfer related technologies to other sectors, such as industries;
• Address the ethical, legal, and social issues (ELSI) that may arise from the project.

Methodologies

• To identifying all the genes that expressed as RNA referred to as Expressed Sequence Tags (ESTs).
• Simply sequencing the whole set of genome that contained all the coding and non-coding sequence, and later assigning different regions in the sequence with functions is called as Sequence Annotation.
• The total DNA from a cell is isolated and converted into random fragments of relatively smaller sizes and cloned in suitable host using specialised vectors.
• The cloning resulted into amplification of each piece of DNA fragment.
• The commonly used vectors are BAC (bacterial artificial chromosomes), and YAC (yeast artificial chromosomes).
• The fragments were sequenced using automated DNA sequencers.
• Specialized computer based programmes were developed for the alignment of the sequences.
• The sequences were subsequently annotated and were assigned to each chromosome.
• The sequence of chromosome 1 was completed only in May 2006.

Automated DNA sequencer 

Salient Features of Human Genome

• The human genome contains 3164.7 million nucleotide bases.
• The average gene consists of 3000 bases with the largest known human gene being dystrophin at 2.4 million bases.

The largest known human gene- dystrophin 

• The total number of genes is estimated at 30,000.
• 9 per cent nucleotide bases are exactly the same in all people.
• The functions are unknown for over 50 per cent of discovered genes.
• Less than 2 per cent of the genome codes for proteins.
• Repeated sequences make up very large portion of the human genome.
• Repetitive sequences are stretches of DNA sequences that are repeated many times.
• Chromosome 1 has most genes (2968), and the Y has the fewest (231).
• Scientists have identified about 1.4 million locations where single base DNA differences (SNPs – single nucleotide polymorphism) occur in humans.

Single nucleotide polymorphism (self-drawn )

Applications of HGP

• All the genes in a genome can be studied together.
• Helps to understand how tens of thousands of genes and proteins work together in interconnected networks.
• Helps to diagnose and treat genetic diseases.