Genetic Engineering, Cloning and Genomics
1
. Introduction
1. Genetic engineering involves transfer or replacement of genes, so also known as recombination DNA technology or gene splicing.
2. Genetic engineering aims at adding, removing or repairing a part of genetic material.
3. Gene splicing is the cutting of DNA molecules at precise points with the help of enzymes.
4. Genetic engineering helps in the rectification of genetic errors.
5. Har Goving Khorana of India is associated with genetic engineering. He synthesized 'gene' artificially in a test tube (1969).
6. First protoplast fusion was done by Harris and Watkins of Oxford using somatic cells of mouse and man.
7. Hybridization by protoplast fusion is called parasexual hybridization.
Recombinant DNA Technology:-
1. The term recombinant DNA refers to the creation of a new association between DNA molecules or segments of DNA molecules from different biological sources.
2. Genetic engineering means manipulation of genes and it depends upon recombinant DNA technology.
3. Genetic engineering has revolutionized genetic studies in higher organisms and is a powerful tool in basic research on microbial genetics.
4. Genetic engineering is providing vast opportunities for improving processes of economic value and treating diseases.
Tools of Genetic Engineering:-
8. Nuclease are enzymes used in genetic engineering that cut, shorten or degrade e.g. restriction endonuclease and restriction exonuclease.
6. Restriction endonuclease is used to cut the plasmid as well as the foreign DNA molecules at specific points.
7. Ligase, another enzyme is used to seal gaps or to join pieces of DNA.
Restriction Endonucleases:-
8. 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 (from the Greek term endon meaning "within";
9. Endonucleases make internal cuts in DNA molecules).
10. Restriction endonucleases are also called as 'molecular scissors' or 'chemical scalpels'.
11. Many endonucleases make random cuts in DNA, but the restriction endonucleases are site-specific.
12. Restriction endonucleases cleave DNA molecules only at specific nucleotide sequences called restriction sites.
13. Different restriction endonucleases are produced by different microorganisms and recognize different nucleotide sequence in DNA.
14. The restriction endoculeases are named by using the first letter of the genus and the first two letters of the species the produces the enzyme.
15. If an enzyme is produced only by a specific strain, a letter designating the strain is appended to the name.
16. The first restriction enzyme identified from a bacterial strain is designated I, the second II, and so on.
17. Thus, restriction endonuclease EcoRI is produced by Escherichia coli strain R Y I3.
18. Restriction enzyme called Eco RI recognizes the sequence
GAATTC
CTTAAG
and cleaves the DNA between G and A on both strands.
19. Restriction endonucleases were discovered in 1970 in Bacteria by Hamilton Smith and Daniel Nathans.
20. Nobel Prize of 1978 for restriction endonuclease technology and their role in genetic engineering was given to Daniel Nathans (USA), Hamilton Smith (USA) and Werner Arber (Switzerland) (BHU 1989).
21. The biological function of restriction endonucleases is to protect the genetic material of bacteria from "invasion" by foreign DNAs, such as DNA molecules from another species or viral DNAs.
22. As a result, restriction endonucleases are sometimes referred to as the immune systems of prokaryotes.
Characteristic 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' GGATC C 3'
3' C CTAGG 5'
Bgl II
"bagel-two"
Bacillus globigi
AG A T C T
T C T A G A
Eco RI
"echo-R-one"
E. coli RY 13
GA A T T C
C T T A A G
Hind III
"hin-D-three"
Haemophilus influenzae Rd
AA 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 hemolyticus
G G CC
C C G G
Hha I
"ha-ha-one"
Haemophilus hemolyticus
G C GC
CG C G
Hpa II
"heap-two"
Haemophilus parainfluenzae
CC G G
G G CC
23. All cleavage sites in the DNA of an organism must be protected form cleavage by the organism's own restriction endonucleases; otherwise the organism would commit 'suicide' by degrading its own DNA.
24. In many cases, this protection of endogenous cleavage sites is accomplished by methylation of one or more nucleotides in each nucleotide sequence that is recognized by the organism's own restriction endonuclease.
25. Methylation occurs rapidly after replication, catalyzed by site-specific methylases produced by the organism.
26. Each restriction endonuclease will cleave a foreign DNA molecule into a fixed number of fragments, the number depending on the number of restriction sites in the particular DNA molecule.
27. An interesting feature of restriction endonucleases is that they commonly recognize DNA sequences that are palindromes that is, nucleotide-pair sequences that read the same forward or backward form a central axis of symmetry.
28. In addition, a useful feature of many restriction nucleases is that they make staggered cuts; that is, they cleave the two strands of a double helix at different points.
29. Other restriction endonucleases cut both strands at the same place and produce blunt ended fragments.
30. Restriction endonuclease Sma I is produced by Serratia marcescens recognizes the sequence
C C C G G G
G G G C C C
producing blunt ends.
31. Because of the palindromic nature of the restriction sites, the staggered cuts produce segments of DNA with complementary single stranded ends.
32. DNA fragments can be rejoined under the appropriate renaturation condition by using the enzyme DNA ligase to reform the missing phosphodiester linkages in each strand.
33. Thus, DNA molecules can be cut into pieces, called restriction fragments and the pieces can be joined together again with DNA ligase.
34. Exonucleas, which remove nucleotide one at a time form the end of the a DNA molecules. These enzymes are less useful in genetic engineering.
35. Polymereses make copies of molecules.
36. Topoisomerases introduce or remove super coils form covalently closed circular DNA.
c-DNA:-
1. c-DNA stands for complementary DNA or copy DNA.
2. c-DNA is a DNA copy of an RNA and is made from m-RNA that codes for a known protein by reverse transcription or Teminism.
3. c-DNA can be formed by RNA dependent DNA polymerase or Reverse Transcriptase.
4. c-DNA is used in a gene cloning technology.
5. c-DNA is also made to use PCR (Polymerase Chain Reaction) to amplify an RNA.
6. 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).
Vector:-
1. Vector in genetic engineering is usually a DNA segment used as a carrier for transferring selected DNA into living cells.
2. A vector may be used to construct recombinant DNA molecules. Some of the example are:
Plasmid vectors
Bacteriophage vectors
Cosmid vectors
BACs (Bacterial Artificial Chromosomes)
YACs (Yeast Artificial Chromosomes) and
MACs (Mammalian Artificial Chromosomes)
Phase P-1 derived artificially chromosome (PACs) are another relatively recently developed class of vector for use in E. coli.
3. Plasmids are extrachromosomal, covalently closed circular double stranded molecules of DNA present in most prokaryotes.
4. Bacteria contain one or more different plasmids; their functions are often unknown.
5. Many plasmids are without vital genes but carry genes for sexuality, antibiotic resistance, virulence etc., bacteria can survive without plasmids.
Plasmid Classification:-
1. Fertility or F plasmids carry tra genes and have no characteristic beyond the ability to promote conjugal transfer of plasmids e.g. F plasmid of Ecoli.
2. Resistance or R plasmids carry genes conferring on the host bacterium resistance to one or more antibacterial agents such as ampicillin, chloramphenicol, Hg e.g. RP4, commonly found in Pseudomonas.
3. Col plasmids code for colicin – protein that kill other bacteria e.g. col E1 of Ecoli
4. Degradative plasmids allow the host bacterium to metabolized unusually molecules such as Toluene and salicylic acid. E.g. TOL of Pseudomons putida
5. Virulence plasmids confers pethogenicity on the host bacterium e.g. Ti plasmids of Agro bacterium tumefaciens which induce crown gall decease in dicot plants, Ri plasmids of Agro bacterium rhizogens which causes hairy root disease in plants.
Some Plasmid-Coded Traits
Trait
Organisms in which Traits is found
Antibiotic resistance
Escherichia coli
Insect toxin synthesis
Bacillus thuringiensis
Nitrogen fixation
Rhizobium sp.
Oil degradation
Pseudomonas sp.
Toxin production
Bacillus anthracis
Tumour formation in plants
Agro-bacterium tumefaciens
6. All plasmids carry replicons pieces of DNA or ORi that have the genetic information required to replicate.
7. Plasmids vary in size from few genes to several hundred.
8. Plasmids range from about 1 kb (kilobase = 100 base pairs) to over 200 kb in size and replicate autonomously.
9. Plasmid pBR 322 was one of the first widely used cloning vectors, it contain both ampicillin and tetracycline resistance genes and having 4363 base pairs it was discovered by Boliver and Rodriguez.
10. Most bacteriophage cloning vectors have been constructed from the phage chromosome.
11. The hybrids between plasmids and the phage chromosome give rise to cosmid (for cos site and plasmid) vectors.
12. Artificial chromosomes like BACs, YACs and MACs are very efficient vectors for eukaryotic gene transfers.
The nomenclature of plasmid cloning vectors:-
The name 'pBR322' conferms with the standards rule for vectors nomenclature.
'p' indicate that this is indeed a plasmid.
'BR' identifies the laboratory in which the vector was originally constructed (BR strand for Boliver and Rodriguez the researchers who developed pBR322.
322 distinguishes this plasmid from other developed in same laboratory (there are also plasmid called pBR325, pBR327, pBR328, etc.).
Tumour-inducing (Ti) Plasmid:-
1. One of the most interesting plasmids is the tumour-inducing (Ti) plasmid of Agrobacterium tumefaciens.
2. Agrobacterium tumefaciens is the causative agent of a common plant disease termed crown gall disease.
3. Agrobacterium tumefaciens is an natural genetic engineered; it infects a wound, and injects a short stretch of DNA called T-DNA into some of the cells around the wound.
4. It is possible to remove the tumour-producing genes that are transferred to the plant and substitute useful genes such as the B. thuringiensis toxin genes.
5. The toxin is naturally produced by the bacterium B. thuringiensis as it forms endospores.
6. The toxin binds to specific receptors in the insect gut, causing gut to dissolve.
7. These useful genes are then transferred and integrated into the plant.
8. When the genes for B. thuringiensis toxin are transferred to a plant, the plant becomes toxic to many insect pests, thus, conferring insect resistance.
Transduction:-
1. Transfer of live genetic material is involved in transduction and sexduction.
2. Transduction is the transfer of bacterial genetic material from one bacterium to another using a phage as the vector.
3. Sexduction is the process in which a fragment of genetic material from one bacterium is carried with the sex-factor F to a second bacterium.
Polymerase Chain Reaction (PCR):-
1. Polymerase Chain Reaction (PCR) was developed by Kary Mullis in 1983 while he was a staff chemist at the Cetus Corporation in California.
2. In 1993, PCR won Kary Mullis the Nobel Prize for chemistry.
3. PCR is nicknamed as 'People Choice Reaction'.
4. PCR is a method for amplifying a specific piece of DNA molecule without the requirement for time-consuming cloning procedures.
5. A direct procedure to copy the gene sequence of interest is called the PCR.
6. PCR starts with double stranded DNA molecule from which millions of identical copies of a select region can be produced.
7. Four ingredients of the PCR are:
(i) the double stranded DNA to be amplified called the target DNA.
(ii) a heat stable DNA polymerase, which work at optimum temperature of 72oC (usually Taq DNA polymerase obtained from the thermophile Thermus aquaticus a bacterium lives in hot water at a temperature 75oC. )
(iii) each of the four nucleotides (dATP, GTP, dCTP, dTTP); and
(iv) small single stranded strands of DNA of about 20 nucleotides called primers.
(v) a primer is short nucleotide sequence that can be extended by DNA polymerase in PCR.
8. An automatic thermal cycler is used in PCR which has a microprocessor controlled temperature cycling.
9. PCR technique has now been automated and is carried out by a specially designed machine.
10. Currently a PCR machine can carry out 25 cycles and amplify DNA 105 times as little as 57 minutes.
11. PCR has revolutionized many aspects of science and medicine because it permits the investigation of minute samples of DNA, PCR is used in the test of HIV.
Nucleic Acid Blotting:-
1. Nucleic acid blotting techniques are employed for the characterization of cloned sequences.
2. Four nucleic acid blotting techniques are Southern blot, Northern blot, Western blot and Southwestern blot.
3. One of the most useful methods developed by Edward Southern for identifying a specific gene is the Southern blot.
4. Southern blotting technique is used for separating DNA fragments and identification of cloned genes.
5. Northern blotting technique can be used to determine whether a cloned gene is transcriptionally active in a given cell or tissue type, this technique uses RNA.
6. Western blotting technique involves hybridization of protein bound to a filter.
7. Southwestern blotting technique is a variant of the Southern blot used to find protein molecules that stick to DNA molecules.
8. Gel electrophoresis and autoradiography are employed in nucleic acid blotting.
Medical Diagnosis of Diseases:-
1. Diagnosis of diseases can be done with DNA probes, short segment of single stranded DNA attached to a radioactive or fluorescent marker.
2. Small oligonucleotides capable of recognizing complementary sequence are known as molecular probes.
3. A molecular probe might be used to find a nucleotide sequence.
4. In Southern blotting, DNA is visualized by DNA probes.
5. DNA probes are used for identification of infectious agents like Salmonella (food poisoning), Staphylococcus (pus forming), hepatitis virus, HIV, etc.
Applications of Recombinant DNA Products
Recombinant Product
Potential Use
Blood-clotting factor VIII
Treatment of haemophilia
Calcitonin
Treatment of osteomalacia
Erythropoietin
Treatment of anaemia
Growth hormone
Growth promotion
Insulin
Treatment of diabetes
Interferons
As antiviral, antitumour and anti-inflammatory agents
Interleuking
Treatment of immune disorders and tumours
Relaxin
Aid to childbirth
Serum albumin
Plasma supplement
Stomatostatin
Treatment of acromegaly
Streptokinase
Anticoagulant
Tissue plasminogen activator
Anticoagulant
Gene Therapy:-
1. The use of bioengineered cells or other biotechnology techniques to treat human genetic disorders is known as gene therapy.
2. Gene therapy is the transfer of normal genes into body cells to correct a genetic defect.
3. Gene therapy can be used to treat genetic diseases like sickle-cell anaemia and Severe Combined Immuno-Deficiency (SCID).
4. SCID is caused by a defect in the gene for the enzyme adenosine deaminase (ADA).
5. SCID patients have no functioning T lymphocytes.
6. SCID patients are treated with the injections of their white blood cells that have been engineered to carry the normal ADA alleles.
2. CLONING
1. Clone is a population of cells or individuals which are genetically identical.
2. The term 'clone' is derived from the Greek word 'Klan' meaning 'twig'.
3. Cloning is the process of producing many identical organisms or cones.
4. Cloning is traditionally done by horticulturists who develop a new plant by just planting a twig from parental plant.
5. There are two types of cloning, gene cloning at molecular level and cloning of organisms.
6. Cloning is meant for preservation of the genotype of the organism.
7. With the death of an organism, a particular genotype is lost.
Microbial Cloning:-
1. Each of the genetically altered or modified microbial cell can be duplicated every time it divides.
2. In just a few days there will be millions of cloned cells, each one canying a copy of the original donated gene.
3. Several improved genetically altered strains of microorganisms can be cloned in large numbers for various applications.
Genetically Engineered Microbes and their Applications
Microbe
Application
Bacillus thuringiensis
Production of Bt toxin
Escherichia coli
Production of human insulin, growth hormone, interferon, etc.
Pseudomonas fluorescence
Prevention of frost damage to plants
Pseudomonas putida
Scavenging of oil spills by digesting hydrocarbons of crude oil.
Rhizobium meliloti
Nitrogen fixation by "nif" gene in cereal crops.
Landmarks in Recombinant DNA Technology
1958 DNA polymerase purified.
1970 A complete gene synthesized in vitro.
Discovery of the enzymes restriction endonuclease and reverse transcriptase.
1972 First recombinant DNA molecules.
1973 Use of plasmid vectors for gene cloning.
1975 Southern blot technique for detecting specific DNA sequences.
1976 Genetech founded, a biotechnology company.
1977 Methods for rapid DNA sequencing.
Discovery of "split genes" and somatostatin synthesized using recombinant DNA.
Genetech produce first human protein (somato statin) in a micro organil.
1978 Human genome library constructed.
1979 Insulin synthesized using recombinant DNA.
First human viral antigen (hepatitis B) cloned.
1982 Commercial production of E. coli genetically engineered human insulin (Eli Lilly & co.). Isolation, cloning and characterization of a human cancer gene (Oncogene ).
1983 Engineered Ti plasmid used to transform plants.
Development of the Polymerase Chain Reaction (PCR) technique.
1988 The first successful production of a genetically engineered staple crop (soyabeans).
Development of the gene gun.
1991 Development of transgenic pigs and goats capable of manufacturing proteins such as human haemoglobin.
First test of gene therapy on human cancer patients.
1994 The Flavr Savr tomato introduced, the first genetically engineered whole food approved for sale.
Fully human monoclonal antibodies produced in genetically engineered mice
1995 Haemophilus influenzae genome sequenced.
1996 Methanococcus jannaschii and Saccharomyces cerevisiae genomes sequenced.
1997 Human clinical trials of antisense drugs and DNA vaccines begun E. coli genome sequenced.
1998 First cloned mammal (the sheep Dolly)
The complete sequence of the nematode Caenorhabditis elegans genome.
2000 Complete sequence of the euchromatic portion of the fruitfly Drosophila melanogaster genome
Complete sequence of the weed Arabidopsis thaliana (Mouse Ear Cress) genome.
2001 International Human Genome Sequencing Consortium and Celera Genomics published the first drafts of the sequence of the human genome.
2002 International Rice Genome Sequencing Project and Syngenta published first draft of the gnomic sequence of two rice subspecies.
Cell Cloning:-
1. Cell cloning is based on the property of totipotency (in plants) or pluriopotency (in animals).
2. Totipotency is the potential ability of a plant cell to grow into a complete plant.
3. Pluriopotency is the potential ability of a cell into develop any type of the cell in the animal body for instance nerve cells or kidney cells or heart cells.
4. Virtually all plants are totipotent but in animals only fertilized egg and stem cells in the embryonic blastocyst are totipotent.
5. Moreover in contrast to animals, there is no real distinction in plants between somatic cells and germ line cells.
Plant Cloning:-
1. Many orchids producing beautiful flowers are cloned plants.
2. Scientists have genetically engineered agronomically important crop plants.
3. Clones of plants are easy to get by vegetative propagation or tissue culture.
4. Rapid production can be achieved using actively dividing meristematic cells.
5. These occur at the root and shoot apices that are growth areas of the plant.
6. For the agriculture, disease, drought, insect pest-resistant as well as herbicide-tolerant crops have been successfully produced by gene manipulation.
7. Genetically Modified Food (GMF) like vitamin A-rich rice (Golden rice) and lysine-rich pulse seeds are now becoming components of human staple food.
Animal Cloning:-
1. Animal cloning is more difficult than plant cloning.
2. This is because animal cells lose their totipotency on reaching the gastrula stage of development.
3. Simian cloning (cloning of monkeys) was camed out by Don Wolf (USA) in 1996 from an eight-cell embryo.
4. ANDI was the first clone monkey by embryo splitting derived its name from a backward abbreviation for 'Inserted DNA'.
5. ANDI was the world's first genetically altered primate produced by inserting a jellyfish gene into the embryo of a rhesus monkey.
6. Ian Wilmut of Roslin Institute, Edinburgh, U.K. has produced a clone of adult lamb named "Dolly" (Feb. 1997).
7. The scientists extracted genetic material from the udder cells of one sheep and implanted it into another sheep's egg after removing its genetic material.
8. The fused cell developed into an embryo which was planted into the uterus of another sheep which acted as surrogate mother.
9. Recent genetic analysis of Dolly's DNA has shown that she is a "chimera", not a perfect clone.
10. Dolly has two genetic mothers as confirmed by the analysis of her mitochondria by Eric Schon and Ian Wilmut in 1999.
11. Dolly has nuclear genes from the ewe who supplied the udder cell an mitochondrial genes from the egg cytoplasm of the second ewe.
12. The Scottish scientists who cloned Dolly have (July 1997) produced Molly and Polly, two lambs clone with a human gene for blood clotting Factor IX.
13. The milk of "Molly" and "Polly" contains Factor IX that can be extracted for us in treating human haemophilia.
14. Steven Stice and James Robl (USA) have developed a technique for genetically customized calves that will be able to produce medicines for humans in their milk.
15. The first cloned calves George and Charlie were born in January 1998.
Human Cloning:-
1. Brigitte Boisslier, a 46-year old French chemist announced the creation of the world's first cloned human baby nicknamed "Eve" (December 2002).
2. Brigitte is the president of the Clonaid human cloning society which believes that man kind was created by extra-terrestrials.
3. Standard DNA profiling of both the baby and mother are not available to confirm the claim made by Brigitte Boisslier.
4. If the baby is a clone, its DNA will match both the nuclear DNA and the mitochondrial DNA of the woman who was said to be cloned.
3. TRANSGENICS
1. A gene that has been introduced into a cell or organism is called a transgene (for transferred gene) to distinguish it from endogenous genes.
2. The organism carrying the introduced foreign gene is said to be transgenic.
3. Thransgenics or transgenic organisms are also called Genetically Modified Organisms (GMOs).
4. Transgenics were first created and expressed in microorganisms, now techniques are available to make transgenic animals and higher plants.
Transgenic Animals:-
1. Two methods are used to produce transgenic animals:
(i) microinjection of DNA into fertilized eggs and
(ii) infection of preimplantation embryos with retroviral vectors.
2. Most of the transgenic animals studied to date were produced by microinjection of DNA into fertilized eggs.
3. Prior to microinjection, the eggs are surgically removed from female parent and fertilized in vitro.
4. The DNA is then microinjected into the male pronucleus of the fertilized egg through a very fine-tipped glass needle.
5. Usually, several hundred to several thousand copies of the gene of interest are injected into each egg.
6. The integration on injected DNA molecules appears to occur at random sites in the genome.
7. 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.
8. Transgenic animals have been genetically engineered with human genes in order to make products that can be used in human medicine.
9. Transgenic cattle (cow, sheep and goat) have been produced with the intention of increased milk yield, as well as, therapeutic human proteins.
10. The transgenic cattle secrete these useful proteins in their milk from where it can be 'harvested'.
11. Transgenic pigs have been given human genes so that their organs carry human antigens.
12. Transgenic pigs organs like heart, kidney, pancreas can be transplanted into humans. Surprisingly, these are not rejected.
13. Transgenic pigs show undesirable side effects of the higher growth hormone levels.
14. Most the female transgenic pigs are sterile, in addition both sexes are lethargic with weak muscles and are highly susceptible to arthritis and ulcers.
Transgenic Plants:-
1. Plants have been genetically manipulated by plant breeders for decades.
2. At present plant breeders can directly modify the DNA of plants; they can quickly add genes from other species to plant genomes by recombinant DNA techniques.
3. Transgenic plants can be produced by several different procedures.
4. One widely used procedure, called microprojectile bombardment, involves shooting DNA-coated tungsten or gold particles into plant cells.
5. Another procedure, called electroporation, uses a short burst of electricity to get DNA into the cell.
6. The most widely used method of generating transgenic plants, at least in dicots, is probably Agrobacterium tumefaciens mediated transformation.
7. The gene coding for the insecticidal protein form Bacillus thuringiensis can be transferred to the cotton plant and this transgenic cotton plant known as genetically modified cotton Bt is resistant to bollworm.
8. The genetic engineering permits the production of transgenic GMO tomato called Flavr Savr.
9. Flavr Savr tomato has much longer and more flavourful shelf-life than conventional tomatoes, because of delayed ripening.
10. Delayed ripening is possible by reducing the amount of cell wall degrading enzyme 'polygalacturonase' responsible for fruit softening.
4. DNA FINGERPRINTING
1. DNA fingerprinting also known genetic fingerprinting was developed by a British geneticist Prof. Alec Jefferys in 1984 at the University of Leivester.
2. A more recent and more sensitive version is known as DNA profiling.
3. DNA fingerprinting was first used in Britain to verify the parentage of an immigrant who had left the country and then wanted to return.
4. Each of us is genetically unique (with the exception of identical twins) and genetic variation could be used to identify individuals, much as conventional fingerprints does.
5. The chromosomes of very human cell contain scattered through their DNA short, highly repeated 15 nucleotide segments called "mini-satellites" or Variable-Number Tandem Repeat (VNTR).
6. The location and number of repeats of any particular minisatellites are so highly variable that no two people are alike.
7. The probability of two unrelated individuals having same pattern of location and repeat number of minisatellites is one in ten billion (in a world population of just over 5.4 billion); no two persons are alike.
8. Restriction Fragment Length Polymorphism (RFLP) distributed throughout human genomes are useful for gene mapping and DNA fingerprints.
9. The basis of DNA fingerprinting is the occurrence of restriction fragment length polymorphism.
10. DNA fingerprint method is very useful for DNA tests for identity and relationships, forensic studies and polymorphism.
Technique for DNA Fingerprinting:-
11. Only a small amount of tissues like blood or semen or skin cells or the hair root follicle is needed of DNA fingerprinting.
12. Typically DNA content of about 100,000 cells or about 1 microgram is sufficient.
13. The procedure of DNA fingerprinting involves the following major steps:
(i) DNA is isolated from the cells in a high-speed refrigerated centrifuge.
(ii) If the sample of DNA is very small, DNA can be amplified by Polymerase Chain Reaction (PCR).
(iii) DNA is then cut up into fragments of different length using restriction enzymes.
(iv) The fragments are separated according to size using gel electrophoresis through an agarose gel. The smaller fragments move faster down the gel than the larger ones.
(v) Double stranded DNA is then split into single stranded DNA using alkaline chemicals.
(vi) 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).
(vii) The nylon sheet is then immersed in a bath and probes or markers that are radioactive synthetic DNA segments of known sequences are added. The probes target a specific nucleotide sequence which is complementary to VNTR sequence and hybridizes them.
(viii) 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 items.
Application of DNA Fingerprinting:-
This technique is now used to:
(i) Identify criminals in forensic laboratories.
(ii) Settle paternity disputes.
(iii) Verify whether a hopeful immigrant is, as he or she claims, really a close relative of already an established resident.
(iv) Identify racial groups to rewrite biological evolution.
5. GENOMICS
1. The term genome has been introduced by Winkler in 1920.
2. Genome refers to one complete copy of the genetic information (DNA) or one complete set of chromosomes (monoploid or haploid) of an organism.
3. The term genomics is relatively new, coined by Thomas Roderick in 1986.
4. Genomics is the sub-discipline of genetics devoted to the mapping, sequencing and functional analysis of genomes.
5. Genomics is subdivided into structural genomics and functional genomics.
6. Structural genomics is the study of genome structure, deals with the complete nucleotide sequences of the organisms.
7. Functional genomics is the study of genome function which includes transcriptome and proteome.
8. Transcriptome is a complete set of RNAs transcribed from a genome.
9. Proteome is a complete set of proteins encoded by a genome and aims at the determination of the structure and functions of all the proteins in living organisms.
10. During the last two decades, the complete DNA sequences of following have been determined:
(i) The genomes of 599 viruses and viroids.
(ii) 205 naturally occurring small intracellular self-replicating DNA molecules called plasmids.
(iii) 185 chromosomes of organelles such as chloroplasts and mitochondria.
(iv) Genomes of 32 eubacteria, 7 archaea, a fungus, a plant and 2 animals.
(v) In addition, two drafts of the sequences of the human genome have been published.
11. The nucleotide sequence of the genomes of two subspecies of rice were published in April 2002.
12. Human body contains 100 million cells of over 260 different kinds.
13. There is a complete set of instruction in each of the cell needed to make a whole new human being.
14. There are 23 different chromosomes containing packed DNA in a haploid set of human genome.
15. Additional DNA is in cell's mitochondria which is inherited from one's mother.
The Human Genome Project:-
1. 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.
2. The goals of this project are:
(i) To map all the human genes
(ii) To construct a detailed physical map of the entire human genome, and
(iii) To determine the nucleotide sequence of all 24 human chromosomes by the year 2005.
3. An international Human Genome Organization (HUGO) was organized to coordinate the efforts of human geneticists around the world.
4. James Watson was the first director of human genome project; after leadership during the first two years of the project, he was replaced by Francis Collins in 1993.
5. Two important scientists associated with human genome are: Francis Collins, director the Human Genome Project and J. Craig Venter, founding president of Celera Genomics.
6. The complete sequencing of the first human chromosome, small chromosome 22, was published in December 1999.
7. The complete sequence of human chromosome 21 followed in May 2000.
8. The first-draft sequences of entire human genome has appeared in the February 15, 2001 issue of Nature and the February 16, 2001 issue of Science.
9. According to present estimate there are about three billion nucleotide base pairs in human genome.
10. The number of genes in human genome is approximately 30,000 to 40, 000.
11. The number of genes in mice genome is almost same as human and nine-tenths of human genes are identical to that of mice.
12. Human being possess more than twice as many as genes in Drosophila.
13. Human beings are 99.9 per cent identical with each other at the DNA level.
14. Every alive human being is exactly the same and even bacteria are our cousins in code.
15. Different human genes vary widely in the length often over thousands of base pairs.
16. While -globin and insulin gene are less than 10 kilobase pairs, the gene responsible for Duchenne Muscular Dystrophy on 'X' chromosome is made up of 2400 kilobase pairs which is probably the longest gene known.
17. Though lily plant produces fewer proteins than human being, it has 18 times more DNA.
18. Only less than 2 per cent of the genome is known to include the exons, the protein coding sequences.
19. Approximately 1 million copies of short 5 to 8 base pair repeated sequence clustered around the centromeres and near the ends of the chromosomes represent 'the junk DNA'.
20. Associated with the Human Genome Project the parallel efforts to obtain gene maps and complete sequence of the genomes of a number of other model organisms, including E. coli. Yeast, Drosophila melanogaster, mouse, etc.
21. Sequencing of the somplete yeast, Saccharomyces cerevisiae, genome was completed in 1996; yeast was the first eukaryotic organism to have its entire genome sequenced.
22. Yeast genome contains an estimated 6,000 genes having 12 million base pairs.
23. In early 1997, the complete 4.7 million base pairs genome with 4000 genes of E. coli was reported.
24. E. coli is the most studied and best understood cellular organism on our planet.
25. Drosophila genome contains 180 million base pairs and 13,000 genes, fewer than the 18,000 genes reported in the nematode Caenorhabditis elegans, however the latter has only 97 million base pairs.
26. Drosophilla Melanogaster (fruit fly) is fondly regarded as Queen of Genetics or Cindrella of Genetics.
Genome of Model Organisms
Organism
No. of Base pairs
No. of genes
Bacteriophage
10 thousand
–
Escherichia coli
4.7 million
4,000
Saccharomyces cerevisiae
12 million
6,000
Caenorhabditis elegans
97 million
18,000
Drosophila melanogaste
180 million
13,000
Human
3 billion
30,000
Lily
106 billion
–
Prospects and Implications of Human Genome:-
1. The genome project is being compared to the discovery of antibiotics.
2. We will soon have details of more than 1200 genes that are responsible for common cardiovascular ailments, endocrine diseases, neurological disorders like Alzheimer's disease, deadly cancers and many more.
3. Efforts are in progress to determine genes that will revert cancerous cells to normal.
4. The human genome sequencing not only holds promise for a healthier living, it also holds the prospects of cast database of knowledge about designer drugs, genetically modified diets and finally our genetic identity.
6. GENE LIBRARIES AND GENE BANKS
1. A gene library is a collection of gene clones that contains all the DNA present in some source.
2. If the original source of the DNA was original DNA from a living organism, then the library seeks to include clones of all that DNA; it is called a genomic gene library.
3. Gene libraries can also be created using RNA.
4. If a gene library is created by enzymatic copying of RNA by reverse transcriptase (RNA-dependent DNA polymerase), it would be called c-DNA library.
5. A gene bank is repository of clones of known DNA fragments, genes, gene maps, seeds, spores, frozen sperms or eggs or embryos.
6. These are stored for possible use in genetic engineering and breeding experiments where species have become extinct.
7. Gene banks can be used increasingly as the rate of extinction increases, depleting the Earth's biodiversity and genetic variety.
8. If the function of the gene is unknown, its nucleotide sequence can be compared with thousands of gene sequences stored in three large computer gene banks, one is Germany, a second in Japan and a third in the United States.
9. Arobidopsis thalina (cruciferae family) is used as a Model plant for genetic studies, since it has the smallest genome among higher plants further its genome has a low amount of repetitive DNA. The genomes has 130 106 bp and estimated 26000 genes.
10. C. elegans is a free living nematode. It is the first multicellular animal whose genome was completely sequenced
11. Many human disease networks are highly conserved in fruitfly. Thus this insect diseases, including neurological disorders.
12. Lesch Nyhan Syndorme is X linked disorder characterized by Mental retardation & bizarre behavior of affected children, including self mutilation. The syndrome is caused by mutations in the gene for hypoxanthine guanine phosphoribosyl transferees (HPRT).
