Chapter 27 Molecular Basis of Inheritance Part 2 by TEACHING CARE online coaching classes
(i) Recombinant DNA technology
- Definition : Genetic engineering, a kind of biotechnology, is the latest branch in applied genetics dealing the alteration of the genetic make up of cells by deliberate and artificial means. Genetic engineering involves transfer or replacement of genes, so also known as recombination DNA technology or gene splicing.
- Tools of genetic engineering : Two enzymes used in genetic engineering are restriction endonuclease and R.E. is used to cut the plasmid as well as the foreign DNA molecules of specific points while ligase is used to seal gaps or to join bits of DNA.
The ability to clone and sequence essentially any gene or other DNA sequence of interest from any species depends on a special class of enzymes called restriction endonucleases. Restriction endonucleases are also called as molecular scissors or ‘chemical scalpels’. Restriction endonucleases cleave DNA molecules only at specific nucleotide sequence called restriction sites. The first restriction enzyme identified from a bacterial strain is designated I, the second II and so on, thus, restriction endonuclease EcoRI is produced by Escherichia coli strain RY
- Restriction enzyme called EcoRI recognizes the sequence G ¯A AT T C
and cleaves the DNA between G and A
on both strands. Restriction nucleases make staggered cuts; that is, they cleave the two strands of a double helix at different joints and blunt ended fragments; that is, they cut both strands at same place.
Characteristics of some restriction endonucleases
|Enzyme name||Pronunciation||Organism in which enzyme is found||Recognition sequence and position of cut|
|Bam HI||“bam-H-one”||Bacillus amyloliquefaciens H||5¢ G¯GAT C C 3¢|
3¢ C C TAG G 5¢
|Bgl II||“bagel-two”||Bacillus globigi||A¯G A T C T|
T C T A GA
|Eco RI||“echo-R-one”||E. coli RY13||G¯ A A T T C|
C T T A AG
|Hae II||“hay-two”||Haemophilus aegyptius||R G C G C¯Y|
YC G C G R
|Hind III||“hin-D-three”||Haemophilus influenzae Rd||A¯ A G C T T|
T T C G A A
|Pst I||“P-S-T-one”||Providencia stuartii||C T G C A¯G|
G A C G T C
|Sma I||“sma-one”||Serratia marcescens||C C C ¯ G G G|
G G G C C C
|Hae III||“hay-three”||Haemophilus aegyptius||G G ¯ C C|
C C G G
|Hha I||“ha-ha-one”||Haemophilus hemolyticus||G C G ¯C|
CG C G
|Hpa II||“hepa-two”||Haemophilus parainfluenzae||C¯ C G G|
G G CC
- Steps of recombinant DNA technology
- Isolating a useful DNA segment from the donor
- Splicing it into a suitable vector under conditions to ensure that each vector receives no more than one DNA
- Producing of multiple copies of his recombinant
- Inserting this altered DNA into a recipient
- Screening of the transformed
- Vectors : Vector in genetic engineering is usually a DNA segment used as a carrier for transferring selected DNA into living Which are as follows
- Plasmid : Plasmid are extrachromosomal, closed circular double stranded molecules of DNA present in most eukaryotes. All plasmid carry replicons pieces of DNA that have the genetic information required to replicate. Plasmid pBR 322 was one of the first widely used cloning vectors, it contain both ampicillin and tetracycline resistance
- Phage : It is constructed from the phage l chromosomes and acts as bacteriophage cloning
- Cosmid : The hybrids between plasmid and the phage l chromosome give rise to cosmid Beside all these there are artificial chromosomes like
BACs (Bacterial Artificial chromosomes) YACs (Yeast Artificial chromosomes)
MACs (Mammalian Artificial chromosomes) are very efficient vectors for eukaryotic gene transfers.
- Application of recombinant DNA technology : The technique of recombinant DNA can be employed in the following
- It can be used to elucidate molecular events in the biological process such as cellular differentiation and The same can be used for making gene maps with precision.
- In biochemical and pharmaceutical industry, by engineering genes, useful chemical compounds can be produced cheaply and efficiently which is shown in
Applications of recombinant DNA products
|Medically useful recombinant products||Applications|
|Human insulin||Treatment of insulin-dependent diabetes|
|Human growth hormone||Replacement of missing hormone in short stature people|
|Calcitonin||Treatment of rickets|
|Chronic gonadotropin||Treatment of infertility|
|Blood clotting factor VIII/IX||Replacement of clotting factor missing in patients with Haemophilia A/B|
|Tissue plasminogen activator||Dissolving blood clots after heart attacks and strokes|
|Erythropoitin||Stimulation of the formation of erythrocytes (RBCs) for patients suffering from anaemia during kidney dialysis or side effects of AIDS patients treated by drugs|
|Platelet derived growth factor||Stimulation of wound healing|
|Interferon||Treatment of pathogenic viral infections, cancer|
|Interleukins||Enhancement of action of immune system|
|Vaccines||Prevention of infectious diseases such as hepatitis B, herpes, influenza, pertussis, meningitis, etc.|
- Cloning : Cloning is the process of producing many identical organisms or clones. In this process nucleus of ovum (n) is removed and replaced by nucleus of diploid cell of same Now the egg with 2n nucleus is transferred to the uterus of mother to have normal pregnancy and delivers clone of itself.
Examples of organism cloning
- Cloning of sheep was done by Ian Wilmut (1995) of Roslin Institute, Edinberg U.K. and normal healthy lamb (DOLLY) was born in Feb, 1996. This lamb was exactly similar to her mother.
- The first cloned calves George and Charlie were born in January
- ANDI was the world’s first genetically altered primate produced by inserting a jelly fish gene into the embryo of a rhesus monkey.
- Scientist at Scotland cloned POLLY and MOLLY. Unlike Dolly, polly and molly were transgenic (they carried human protein gene) polly and molly were born in july 1997.
- Brigitte Boissliar, a 46-year old french chemist announced the creation of the world’s first cloned human boby nicknamed “Eve” (December 2002).
- Polymerase chain reaction (PCR) : It was developed by Kary Mullis in 1983 and won Nobel prize in PCR is a method for amplifying a specific piece of DNA molecule without the requirement for time- consuming cloning procedure. This process require Target DNA, a heat stable DNA polymerase, which work at optimum temperature of 70°C usually Taq DNA and four types of nucleotides with small single stranded strands of DNA of about 20 nucleotide called primers, produce multiple copy of desired DNA.
Fig : DNA amplification by PCR
(iv) Gene libraries and gene banks
- Gene libraries : A gene library is a collection of gene clones that contains all the DNA present in some source. If the original source of the DNA was original DNA from a living organism, then the library seek to include clones of all that DNA, it is called a genomic gene Gene libraries can also be created by using RNA.
- cDNA : If a gene library is created by enzymatic copying of RNA by reverse transcriptase (RNA-dependent DNA polymerase), it would be called c-DNA c-DNA stands for complimentary DNA or copy DNA. c-DNA is made to use PCR to amplify an RNA. PCR does not work on RNA, so one can copy it to DNA using reverse transcriptase and then PCR amplify the c-DNA; this is called RT-PCR (reverse transcriptase PCR).
- Gene bank : A gene bank is repository of clones of known DNA fragments, genes, gene maps, seeds, spores, frozen sperms or eggs or These are stored for possible use in genetic engineering and breeding experiment where species have become extinct.
- DNA finger printing : Alec Jeffreys et al (1985) developed the procedure of genetic analysis and forensic medicine, called DNA finger printing. It is individual specific DNA identification which is made possible by the finding that no two people are likely to have the same number of copies of repetitive DNA sequences of the It is also known as DNA profiling. The chromosomes of every human cell contain scattered through their DNA short, highly repeated 15 nucleotide segments called “mini-satellites” or variable-number Tandem Repeat (VNTR).
- Technique for DNA fingerprinting
- Only a small amount tissues like blood or semen or skin cells or the hair root follicle is needed for DNA
- Typically DNA content of about 100,000 cells or about 1 microgram is
- The procedure of DNA fingerprinting involves the following major steps :
- DNA is isolated from the cells in a high-speed refrigerated
- If the sample of DNA is very small, DNA can be amplified by Polymerase Chain Reaction (PCR).
- DNA is then cut up into fragments of different length using restriction enzymes.
- The fragments are separated according to size using gel electrophoresis through an agarose ge The smaller fragments move faster down the gel than the larger ones.
- Double stranded DNA is then split into single stranded DNA using alkaline
- These separated DNA sequences are transferred to a nylon or nitrocellulose sheet placed over the gel. This is called ‘Southern Blotting’ (after Edward Southern, who first developed this method in 1975).
- The nylon sheet is then immersed in a bath and probes or makers that are radioactive synthetic DNA segments of known sequences are added. The probes target a specific nucleotide sequence which is complementary to VNTR sequences and hybridizes
- Finally, X-ray film is exposed to the nylon sheet containing radioactive probes. Dark bands develop at the probe sites which resemble the bar codes used by grocery store scanners to identify
- Applications of DNA fingerprinting
This technique is now used to :
- Identify criminals in forensic laboratories.
- Settle paternity disputes.
- Verify whether a hopeful immigrant is, as he or she claims, really a close relative of already an established
- Identify racial groups to rewrite biological
- Gene therapy : The use of bioengineered cells or other biotechnology techniques to treat human genetic disorders is known as gene therapy. Gene therapy is the transfer of normal genes into body cells to correct a genetic It can be used to treat genetic diseases like sickle-cell anaemia and Severe Combined Immuno Deficiency (SCID). It (SCID) is caused by a defect in the gene for the enzyme adenosine deaminase (ADA). SCID patients have no functioning T lymphocytes and one treated with the injections of their white blood cells that have been engineered to carry the normal ADA alleles.
- Transgenics : A gene that has been introduced into a cell or organism is called a transgene (for transferred gene) to distinguish it from endogenous The animal carrying the introduced foreign gene is said to be transgenic animal and the possessor called Genetically Modified Organisms (GMOs). Most of the transgenic animals studied to date were produced by microinjection of DNA into fertilized eggs. Prior to microinjection, the eggs are surgically removed from female parent and fertilized in vitro then DNA is microinjected into the male pronucleus of the fertilized egg through a very fine-tipped glass needle. The integration of injected DNA molecules appears to occur at random sites in the genome.
The first transgenic animal produced was the ‘supermouse’ by the incorporation of the gene for human growth hormone by Richard Palmiter and Ralph Brinster in 1981.
- Genomics and human genome project : The term genome has been introduced by Winkler in 1920 and the genomics is relatively new, coined by Thomas Rodericks in 1986. Genomics is the subdiscipline of genetics devoted to the mapping, sequencing and functional analysis of genomes. Genomics is subdivided into following types:
- Structural genomics : It is the study of genome structure deals with the complete nucleotide sequences of the
- Functional genomics : It is the study of genome function which includes transcriptome and proteome. Transcriptome is a complete set of RNAs transcribed from a genome while proteome is a complete set of proteins encoded by a genome and aims the determination of the structure and function of all the proteins in living The human genome project, sometimes called “biology’s moon shot”, was launched on october 1, 1990 for sequencing the entire human genome of 2.75 billion (2.75 ´ 109 or 2750000 bp or 2750000 kilobase pairs or 2750 megabase pairs) nucleotide pairs.
Two important scientist associated with human genome are Francis Collins, director of the Human Genome Project and J. Craig Venter, founding president of Celera genomics. The complete sequencing of the first human chromosome, small chromosome 22, was published in December 1999.
Genome of Model organisms
|S. No.||Organism||No. of base pair||No. of genes|
|(2)||E. coli||4.7 million||4000|
|(3)||Saccharomyces cerevisiae||12 million||6000|
|(4)||Caenorhabditis elegans||97 million||18,000|
|(5)||Drosophila melanogaster||180 million||13,000|
Prospects and implications of human genome :
- The genome project is being compared to the discovery of
- Efforts are in progress to determine genes that will revert cancerous cells to
- The human genome sequencing not only holds promise for a healthier It also holds the prospects of vast database of knowledge about designer drugs, genetically modified diets and finally our genetic identity.
- Pallindromic DNA is a segment of DNA in which the base pair sequence reads the same in both directions from a point of
- Western blotting is the technique used to detect specific
- Northern blotting is the technique used to blot transfer of
- Recombinant DNA is also called chimeric
- Eli Lily (American company) in 1983 produced genetically engineered insulin called humulin with the help of coli plasmid clone.
- DNA foot printing : It determines the location and lengths of binding sites of various proteins that bind to
- Hargovind Khorana is associated with genetic He synthesized ‘gene’ artificially in a test tube (1969).
- Polymerase Chain Reaction (PCR) was developed by Kary Mullis in 1983 and got Nobel prize for
- Southern blotting technique is used for separating DNA fragments and identification of cloned
- Gel electrophoresis and autoradiography are employed in nucleic and
- Delayed ripening is possible by reducing the amount of cell wall degrading enzyme ‘Polygalacturonase’ responsible for fruit