- 1 DNA Fingerprinting – Purpose, Procedure, and How It’s Used
- 1.1 Properties of Alleles
- 1.2 Explanation of the Properties of Alleles
- 1.3 Multiple Alleles
- 1.4 Isoallele
- 1.5 Transposable Genetic Element or Transposon
- 1.6 Transposable Genetic Elements in Bacteria
- 1.7 Ac-DS Elements in Maize
- 1.8 Human Genome Project
- 1.9 Progression of the Human Genome Project
- 1.10 DNA Fingerprinting
- 1.11 Methods in Preparation of DNA Fingerprint
- 1.12 Utility of DNA Fingerprinting
Microbiology is one of the Biology Topics that involves the study of microorganisms, including bacteria, viruses, and fungi.
DNA Fingerprinting – Purpose, Procedure, and How It’s Used
Inheritance of Mendelian features is controlled by the particulate factors that are now called genes. As per Mendelian concepts, the characters may be manifested as alternative or contrasting features for which there are s different factors. This alternative as well as contrasting factors are known as alleles. The geneticists consider the contrasting features in terms of one normal or wild-type feature. The allelic forms of a gene are developed in nature by mutation and they may exhibit different properties in terms of expression. The behaviour of the allelic forms of a gene and their relation with the development of alternative features of a character is very important to study in the science of inheritance.
Mendel observed two varieties of pea plants: one showing round seed and the other showing wrinkled seed. Therefore, the round seed character and wrinkled seed character are contrasting with each other and Mendel predicted that these alternative features of the same character (shape of the seed) are determined by particulate factors of different natures. The expression of a feature as it appears externally may be called the phenotype, whereas due to specific internal makeup, the external appearance of a character is possible and this internal makeup is known as the genotype of the organism.
For the expression of a character, Mendel proposed there should be a pair of genes either in the same form or in alternative forms. The alternative forms of the same gene are the alleles and they may vary with regard to their power of expression. One may be dominant over the other and in heterozygous conditions when a pair of alleles are present the dominant allele will be expressed while the recessive one will remain suppressed. The recessive form of an allele for its expression needs a homozygous condition. In the present case, the round seed feature is dominant over the wrinkled seed feature.
If the gene for the round feature is denoted as R, then its recessive form may be denoted by r. The gene for the round feature being dominant is dominant over r, the gene for the wrinkled feature. Therefore, round features may be expressed both in RR homozygous and Rr heterozygous conditions. Whereas the wrinkled feature may only be expressed in the rr homozygous condition. In certain cases, alleles of a pair may be equally potential for expression in their heterozygous condition. Such alleles are known as codominant, alleles.
In the case of the human MN blood group is controlled by codominant alleles. Blood group MN is expressed in three phenotypes M, N, and MN. M blood type is due to the homozygous condition of gene LM, N blood type is expressed by the homozygous condition of gene LN, whereas the MN blood type is expressed in the heterozygous condition of the two genes as LMLN. In the case of codominance both the alleles are expressed in the heterozygote phenotype. In certain cases neither of the alleles is dominant or they are codominant, but they exhibit some intermediate in heterozygote condition.
In the wild type, allele or normal allele is said to be incompletely dominant in such cases. In the evening primrose flower colour may be red and white, when red is the normal wild type feature. But when a red-flowered plant is crossed with a white-flowered plant the progeny from such a cross give only a pink flower which is an intermediate expression of red and white. Hence, the allele determining the red colour in this plant is incompletely dominant over the allele determining the white color of the flower. F1 hybrid plant if allowed to self-pollinate then the red, pink, and white progeny will appear in 1 : 2 : 1 ratio.
Alleles of a gene are produced by mutation when the genetic structure is altered giving a new form to the normal gene. Thus the altered gene fails to give the normal expression for a feature. The altered feature in this case is found to less adaptive and harmful to some extent to the organism that possesses the new form of the gene. The number of alleles for a particular gene is very important with respect to variation in the expression of a character in the individuals of a species. Whatever may be the number of alleles of a gene, they are attributed with some properties.
Properties of Alleles
Alleles of a gene usually exhibit the following properties.
- Alleles appear as alternative forms of the same gene controlling the expression of the same character. Therefore, different alleles of a gene are related to different features of the same character.
- The alleles of a gene occupy the same position in the homologous chromosomes.
- In diploid organisms never more than a pair of alleles may be present.
- An allele of a gene may appear by mutation under the influence of some external factor and an allele is considered in terms of its normal or wild-type allele.
- An allele of a pair may exhibit different power of expression and thus an allele may be dominant, semi-dominant, codominant, incompletely dominant, or recessive in nature.
- Inter-allelic recombination does not occur and alleles do not show complementation.
- The pair of alleles present in an organism for a character gets segregated during gamete formation.
- Allelic forms of a gene may be present in a homozygous, heterozygous, or hemizygous state in an organism.
- Alleles contribute to the development of variation in nature.
- In contrast to the normal allele, most of the time alleles are harmful to the organism, and in extreme cases, an allele may be lethal.
- Interallelic interaction sometimes gives intermediate expression and often develops hybrid vigour.
- A natural population devoid of all evolutionary forces shows a constant frequency of the alleles generation after generation.
Explanation of the Properties of Alleles
(a) Alleles as alternative forms of the same gene promoting expression of contrasting features of the same character:
A gene when mutated forms an allele and thus the newly formed counterpart of the gene becomes an alternative form of the pre-existing one. The newly developed gene being the alternative form may give a new feature in relation to the pre-existing feature. The pre-existing feature and the newly developed feature are contrasting in appearance and are related to one specific character of the organism. One example may be cited in relation to this. In humans, RBC cells contain normal hemoglobin which is known as HbA. A mutation in the gene-forming beta polypeptide of hemoglobin may result in the production of defective hemoglobin which is known as HbS. When a person becomes homozygous for HbS producing gene, he develops sickle cell anaemia. In this case, the affected person suffers from severe anoxia in low oxygen concentration in the environment.
Sickling of RBC leading to disruption of peripheral circulation may lead to such a condition. HbA and HbS are contrasting by nature and function. Another example may also be given in this regard. Albinism in man is a defective condition when the affected person fails to synthesize melanin in their body and therefore, the affected persons are albino without any pigment on the skin. Such affected individuals face many difficulties in leading a normal life in nature. The normal individual with melanin in the skin appears contrasting by phenotypic features compared to an albino individual. The gene for which the albino condition develops is recessive to its normal allele capable of producing melanin and the normal gene is dominant over the mutant allele for albinism. The normal gene may because of its dominant nature may be designated as A’ and the mutant allele being recessive is designated as ‘a’. Therefore, A and a are alternative forms of the same gene affecting the same character in humans.
(b) Alleles of a gene occupy the same position on the homologous Chromosome:
After the proposition of the chromosome theory of inheritance by Sutton and Bovery in 1903, scientists have been convinced by the concepts of the chromosomal location of the genes or Mendelian factors. Following this, the discovery of linkage by Morgan again gave a solid foundation to the specific arrangement of the genes over a chromosome. The theory also states that the genes are linearly arranged on the chromosome having a specific location and two genes present over a particular chromosome exhibit a defined linkage relationship. On the basis of these concepts we now emphatically say that the allelic position over the identical homologous chromosomes is fixed and their segregation pattern also follows definite principle. This specific location of the allelic counterparts of a gene has further been supported by the linkage mapping of many genes of different diploid organisms.
(c) Alleles can never be more than two in number in an organism:
The diploid organisms contain two sets of chromosomes in their body cells. In such conditions, each chromosome remains in pairs and therefore, the genes present over a particular chromosome also are present in pairs. The presence of such allelic pairs of a specific gene represents the normal condition in the individual. A pair of genes in the allelic series may represent either homozygous or heterozygous conditions. Two genes of the allelic pair when become identical it is known as homozygous condition and being alternative in forms the condition is known as heterozygous condition. In consideration of pigment production in the skin stated above AA or aa represents the homozygous condition and Aa represents the heterozygous condition.
(d) Alleles appear by mutation:
Already stated above the alternative form of a gene may appear only by mutation. Normally a mutation appears some influence from the external side is necessary over the genetic material in the cell is required. In this regard, the mutation causing a change in the base sequence in DNA may be cited here in consideration of the development of HbS and HbA. In this mutation, the protein is altered by the substitution of one amino acid, i.e., valine for glutamic acid at a particular position of the p polypeptide chain of the haemoglobin which is formed by two alpha polypeptide chains and two beta polypeptide chains. However, for resulting this amino acid substitution a transversion from A=T to T=A is required and this is a point mutation.
(e) Alleles having different power of expression:
The example of alleles having different power of expression is clearly evident from Mendelian monohybrid and dihybrid experiments. To consider one monohybrid experiment of Mendel we may cite the cross between pure breeding tall pea plant and dwarf variety pea plant. In this cross, all the F1 progeny appeared as Tall, but when these Fx tall plants were allowed to self-pollinate they produced tall and dwarf plants in 3 : 1 ratio. This indicates that the tall feature is dominant over the dwarf feature and therefore, the gene-controlling tall feature in plants is also dominant over the gene-controlling dwarf feature.
In all the Mendelian crosses in consideration of seven different characters Mendel observed this property of the contrasting features of the seven characters in pea plants. Similar allelic behaviour has also been found in many cases of animals and plants. Not only one allele may be overpowering its allele for its expression (as dominant genes), but there are also examples where two alleles may be equally potential for their expression, and because of this, the heterozygotes express both the effects of the allelic genes. The best example is heterozygote with sickle cell trait in which the individual may produce both types of haemoglobins (HbA and HbS) almost in equal proportion.
(f) Alleles do not recombine and they do not show complementation:
In diploid organisms during meiotic cell division exchange of parts between the homologous chromosomes occurs as a result of crossing over and through this phenomenon, recombination of genes of a chromosome may occur. But crossing over always occurs at a point between two genes on a chromosome and therefore, the phenomenon is intergenic. Hence, the exchange of parts at a point between, the gene segment is either impossible or has no effect on the phenotype of the organism. At the same time a cis arrangement for two alleles over a chromosome is unimaginable and therefore, cis or trans test as suggested for different cistrons is also impracticable. In the trans arrangement, the alleles cannot respond to give wild-type expression which means that alleles do not complement.
(g) Alleles show segregation during gamete formation:
The Diploid organisms mostly reproduce by producing gametes that receive a haploid set of chromosomes from the parental organisms. During gamete formation, the homologous chromosomes are separated from each other and they go into different gametes. Thus during gametogenesis, the genes present over two homologues of an organism get segregated from each other, in other words, allelic pair during gametogenesis are segregated so that each of them may be a part of each of the gametes. The offspring will have both red and white hair (RW). The offspring are heterozygous and called “roan”.
(h) Alleles contribute to the development of variation in organisms:
Alleles in many cases in heterozygous condition gives superiority compared to the homozygotes for the alleles under consideration. This superiority is called heterosis or hybrid vigour.
(i) Alleles are mostly harmful to the organism:
An allele of the gene being a mutant form of its normal counterpart contains comparatively less adaptive value and therefore, to some extent is harmful to the organism. The adaptive value for the normal gene is full and it has achieved stability in the environment therefore, a mutant form of the gene gets less fitness in nature. However, in extreme cases, a mutant gene may be completely lethal having no adaptive value.
(j) Alleles in the absence of evolutionary forces maintains constant frequency in the population:
As per Hardy Weinberg’s principle allelic frequency for a particular gene should remain constant generation after generation provided the population is free from mutation, selection, migration, genetic drift, etc. The stability of a species’ population depends on the frequency distribution of the alleles. However, change in allelic frequency due to evolutionary forces leads to the development of new species from pre-existing ones.
When the number of alleles becomes more than two, the alleles are known as multiple alleles. As the allele number becomes more than two they may produce more than two phenotypic features.
Coat Colour in Rodents
For coat colour variation in rabbits, as many as four alleles could be detected for the development of four different coat colour namely agouti, chindhilla, Himalayan, and albino. The agouti or full colour is the colour pattern in which the coat colour appears as grey. The chinchilla appears as a spotted coat over the grey background, whereas in a Himalayan pattern, only the extremities of the body become black in colour. The albino condition results in a completely white body.
It has been observed that the normal coat colour of rabbits is developed due to a gene called C, chinchilla is due to Cch, Himalayans is due to Ch and albino is due to c. The genes are allelic in nature with a relationship as indicated below C > Cch > Ch > c. Therefore, the presence of C, Cch, and Ch even in heterozygous conditions contributes to agouti, chinchilla, and Himalayan coat colour respectively. When a chinchilla rabbit is crossed with a Himalayan rabbit all the F1 progeny will appear as hybrid indicating the dominance of the gene of chinchilla over that of Himalayan therefore, when the F1 hybrids are allowed to interbreed chinchilla and Himalayan Rabbit in 3 : 1 ratio.
In some cases, multiple alleles act at the same phenotypic range in such a way that the difference between the genes in their expression cannot be easily identified. Such multiple alleles are known as iso alleles. When the iso alleles lead to a phenotypic expression similar to wild-type features, they are called normal or wild-type iso alleles. On the other hand, when the expression of iso alleles appears to be identical to the mutant phenotype, they are called mutant iso alleles.
In Drosophila normal eye colour is red and the feature is dominant in nature. A recessive mutation of the wild-type gene results in a completely white eye. This white feature of the eye depends on the minimum amount of pigment in the eye relative to that in the red eye. However, in Drosophila such eye colours are available that may neither be called typical red as well as typical white. The variations related to eye features are related to variations in the amount of pigment in the eye. Such mutations related to the eye colours of the flies are alleles of the same gene and they act in the same phenotypic range. Therefore they are called iso alleles. Some examples of two types of iso alleles related to the eye colour of Drosophila may be shown in the following table.
The relative amount of eye pigment in case of different eye mutations:
|Mutation||Genotype||Relative Amount of Eye Pigment|
|Canton S wild||w+C/w+C||1.0546|
|Graaf Renet wild||w+G/w+G||1.2548|
Transposable Genetic Element or Transposon
Genes involved in the expression of phenotypic features in living organisms are primarily known to be present in the genome at some fixed position. DNA is the genetic material that holds the genes over it. When a DNA molecule represents a stretch of polynucleotides, a segment of this stretch of polynucleotides acts as a genetic unit. This unit is nothing but a sequence of nucleotides of DNA. This sequence of DNA occupies a specific position over the DNA molecule. In eukaryotes, the DNA molecule is present in the chromosome. Hence, the genes are also present over chromosomes at specific locations.
With the advancement of knowledge in Genetics some scientists found that certain genetic elements in living organisms may change their positions. The genetic elements were called as transposable Genetic Elements. Sometimes they are also called as jumping genes. By changing their position one transposable element may affect the expression of a character and they are therefore, in many cases very important in shaping the life of organisms. Change of position of the transposable elements may involve transfer from one position to the other in the same chromosome or transfer from even one chrome to another.
These sequences are found in all types of organisms are they are structurally and functionally diverse. In the genome, about 40% of regions represent transposable genetic elements. The other name of these elements is transposons. Transposons may be categorized into three types based on some general features as cut and paste transposon, replicative transposon, and retrotransposon. Cut and paste transposon is the sequence that is excised out from one position of the genome and then inserted at the other side of the same genome. On the other hand, a replicative transposon represents a sequence that is first replicated and then the new copy of inserted at some other site in the genome. With this, the original transposon remains at the site and a new copy of it is inserted in the genome. The retrotransposon is the DNA sequence that is formed by reverse transcription of an RNA sequence and then is inserted in the genome. Examples of different types of transposons may be presented in the following table.
Different types of transposons as found in living organisms:
|Type of Transposons||Example||Occurrence|
|Cut and paste transposon||IS elements||Bacteria|
|Replicative transposon||Tn 3 element||Bacteria|
Transposable genetic elements were first discovered in eukaryotes (Maize) by Barbara McClintock in 1940, though they are present in all organisms starting from bacteria to man. The elements may broadly be divisible into three categories namely, cur and paste type transposons, replicative transposons, and retrotransposons. The cut-and-paste type transposons represent the genetic element that being excised out from one opposition of the genome is inserted into another site of the same genome. The second category transposon mans the element that is first replicated to form its own copy and that is inserted into the genome at other sites. In this transposition, one copy of the element remains at its original site. Whereas, the retrotransposon represents a copy of DNA that is produced from one RNA with the help of reverse transcriptase for its transposition into a genome.
Transposable Genetic Elements in Bacteria
In bacteria, there are three types of transposable genetic elements namely insertion sequence or IS elements, composite transposons, and Tn-3-like elements. The study of transposable elements at the molecular level was first carried out in bacteria. Among these three transposable elements, IS element is the simplest and carries a gene producing one protein helpful for its transposition.
IS elements are small compact and simply organized transposable elements of the bacterial cell. Typically an IS element contains fewer than 2500 base pairs containing a single gene that may promote or regulates its transposition. These elements may be inserted in many positions of the bacterial chromosome and plasmids. IS element was first observed in some lac-mutants of E. coli cells. These mutations had an unusual property of reverting to wild type at a higher rate. Molecular analysis showed that the revertants were developed due to the excision of a DNA sequence from the adjacent sequence of the lac operon.
There are many different types of IS elements. The smallest among them is ISI which is composed of 786 nucleotides. By structure, IS element contains shot identical sequence at the ends and the sequence at the two ends is inverted to each other. Hence, these are called terminal inverted repeats. The length of these repeats ranges from 9040 bp. The terminal repeats play an important role in transposition and when there appear mutations at these sites the transposon usually loses the ability to move.
IS element may be transposed from one position to the other in the genome by the activity of an enzyme known as transposase synthesized by the gene possessed by the IS element. With the activity of this enzyme the IS element is excised out from the DNA and inserted in a new position of the same DNA molecule or there. During insertion, the IS element carries out a short duplication of the sequence at the site of insertion and then it is inserted between the duplicated sequence. After insertion, one copy of the duplication sequence remains on each side of the IS element. The short sequence that is duplicated forms a direct repeat and this is known as target site duplication.
The majority of IS elements are between 0.7 and 1.8 kb in size and the termini tend to be 10 to base pairs in length with perfect or nearly perfect repeats. This sequence also tends to have RNA termination signals as well as nonsense codons in all three reading frames and are, therefore, polar. Typically they encode one large open reading frame of 300 to 400 amino acids and by definition, the protein encoded by this reading frame is involved in the transposition event. Two exceptions to the size range given above should be noted: the first, is 5.7 kb, and the other, IS101, is a scant 0.2 kb in size. Although there are exceptions, insertion sequences tend to be present in a small number of copies in the genome. For example, ISI is present in 6 to 10 copies of the E. coli chromosome while IS2 and IS3 are typically present in about five copies.
Transposition of IS element:
IS elements may be transposed from one position to the other in the genome by the activity of an enzyme known as transposase synthesized by the gene possessed by the IS elements. The IS element is first excised out with the help of the enzyme and then inserted in a new position in the same DNA molecule or other. During insertion, the IS element carries out a short duplication of a sequence at the site of insertion and then it is inserted between the duplicated sequence. After insertion, one copy of duplication remains at each side of the IS element. The short sequence that is duplicated forms direct repeats and this is called target site duplication.
When a DNA sequence of any genome is flanked by two IS elements, it becomes capable of transposition from one position to the other. This type of transposon is called composite transposon. Hence, the DNA sequence is captured by two IS elements to represent one composite transposon. It does not contain any gene required for its transposition. Rather the transposase produced by IS element helps transposition the composite transposon.
In most cases, composite transposons are found to carry antibiotic resistance genes. The composite transposon is symbolized by Tn. IS elements in certain cases are found to remain in reverse orientation and sometimes they may be non-identical even. During transposition of composite transposon target site duplication is found to be a classical event. Examples of some composite transposons may be given in the following table.
Some composite transposons of bacterial cells:
|Name of the composite transposon||Size (Bp)||IS element associated||Antibiotic resistance gene associated|
|Tn 9||2500||IS 1||Chloramphenicol resistance|
|Tn 10||9300||IS 10||Tetracyclin resistance|
|Tn 5||5700||IS 50||Kenamycin resistance|
|Tn 903||3100||IS 903||Kenamycin resistance|
This category of transposon is large having no IS element at the ends; rather the terminal ends contain inverted terminal repeats of the size of 30 to 40 bp long. Within the Tn3 element three genes namely tnp A, tnp R, and bla producing transposase, resolvase, and beta-lactamase respectively are present. During transposition, the Tn3 element carries out target site duplication. Transposase and resolvase promote transposition and beta-lactamase gives resistance to ampicillin. This type of transposable element is also called a non-composite transposon.
Tn3 element and transposition:
The tn3 element remains part of the plasmid. During transposition, the plasmid carrying a Tn3 element fuses with another plasmid that does not carry a Tn3 element. This fusion is promoted by the transpose of the Tn3 element and as a result, a co-integrate is formed. Tn3 is a replicative transposon and during co-integrate formation, the Tn3 element is replicated. The extra copy of the Tn3 is integrated with the fused plasmid and the copies of the Tn3 element remain in such a way that one copy of each Tn3 element remains at the junction of the two plasmids. Following this tpn R product resolve promotes site-specific recombination between two copies of Tn3 elements and after this two plasmids will be formed each carrying one Tn3 element.
TE in eukaryotes:
In eukaryotes transposable genetic elements may be divided into classes: Class I type or retrotransposons and Class II type as cut and paste type transposons.
Class I Type Transposon (Retrotransposon):
This type of transposon uses one RNA intermediate for its transposition. Thus the DNA segment is transcribed first and then with the help of reverse transcriptase a copy of DNA is produced which is then inserted in the genome at a new position. The reverse transcriptase required for the synthesis of copy DNA is encoded by the TE itself. Hence, the retrotransposons have the characteristics of retroviruses. These elements can induce mutations by insertion near or within the genes. Around 42% of the human genome is made up of retrotransposons.
Retrotransposons are grouped into three types namely,
- TEs with long terminal repeats (LTRs). It encodes reverse transcriptase similar to retrovirus. Ty1-copia and Ty3-gypsy groups are examples of this type of retrotransposons.
- Long interspersed nuclear elements (LINEs, LINE-Is, or Lis). It encodes reverse transcriptase but lacks LTRs. 17% of the human genome appears to be long interspersed nuclear elements and the human genome contains about 500,000 LINEs.
- Short interspersed nuclear elements. It does not encode reverse transcriptase but is transcribed by RNA polymerase III. They are short DNA sequences with less than 500 bases. As the SINEs do not encode reverse transcriptase and they depend on other mobile elements for transposition. The most common SINEs in primates are called Alu sequences. In humans, SINEs make up about 11% of the genome. There are about 1,500,000 copies of SINES in our genome.
Class II Type Transposon (DNA Transposons):
Class II type transposons are DNA transposons and they do not involve an RNA intermediate for their transposition. They are cut-and-paste type transposons and their transposition is not catalyzed by reverse transcriptase. During transposition, a staggered cut is produced at the target site and the DNA transposon is excised out to be ligated into a new target site. The process involves DNA polymerase and DNA ligase. The insertion sites of DNA transposons may be identified by short direct repeats followed by a series of inverted repeats. Class II TEs comprise less than 2% of the human genome.
Transposable genetic elements may also be classified as autonomous and non-autonomous transposons. Both class I and class II transposons may be categorized into these types. The autonomous transposons move by their own activity, whereas the non-autonomous transposons require another TE for transposition because of the absence of transposase or reverse transcriptase. In maize Ac is an autonomous TE and Ds is the non-autonomous TE and this requires AC for its transposition.
Ac-DS Elements in Maize
These were the first transposable genetic elements of maize discovered by Barbara McClintock in 1948 for which she was awarded the Nobel Prize in 1983. From the analysis of the genetics for the development of seed colour in maize, she predicted that the presence of transposable elements in a chromosome of maize variation in seed colour may be developed. The gens of her selection was present on chromosome number 9 of maize and the genes were C’ a gene that prevents colour formation in the seed, c a recessive gene that promotes colour development on the aleurone, Bz a dominant gene that produces purple aleurone color, bz the recessive gene that produces a dark brown to purple-brown aleurone colour and Ds a genetic location where chromosome breakage occurs. The genetic location of the marker genes on the chromosome was as under.
She performed a cross involving a homozygous female with genotype CCbzbz—meaning the female was homozygous for c and bz genes but lacking Ds and a male with genotype C’C’BzBzdsds. From such a cross the aleurones would have developed with the genotype C’CCBzbzbz—ds. Because of the presence of inhibitor gene C, the akerones should have been colourless. But surprisingly few aleurones developed dark brown coloured sectors over the colourless background. McClintock predicted that somehow the C1 and Bz genes were lost because of chromosome breakage due to the loss of the Ds locus. If breakage occurs at the ds locus then the genotype of the endosperm would be as under fig.
But without a breakage in the chromosome, the genotype would have been:
After crossing with a number of different genetic stocks, she could be able to realize that Ds alone could not induce the breakage. And a second factor, Ac was also necessary. Additional genetic stocks were analyzed by McClintock and she determined that in the presence of Ac, Ds could move locations as well as cause breakages.
She also realized that Ac could move independently but Ds can move only in the presence of AC. Hence, AC is called an autonomous element and DFs are called a non-autonomous element. Therefore, this system is called a two-element system and historically has been called the Ac/Ds system. Therefore, this system is called a two-element system and historically has been called the Ac/Ds system. Ac is 4563 bp in length and contains essential 11-bp inverted repeats at the ends. During transportation, 8 bp direct repeats are generated in the target DNA. On the other hand, the Ds element is a truncated Ac element.
P Element of Drosophila melanogaster
P elements represented a class of transposable genetic elements in fruit flies and have developed much interest among scientists relating to TE and mutations. These are small transposons with terminal 31 bp inverted repeats. The p element during transposition develop 8 bp repeats at the target DNA sequence. The complete p element is autonomous in nature having 2907 bp DNA encoding a functional transposase. Some natural population of flies contains few p elements while others contain as many as 50 p elements in the cell. It is very surprising that flies captured before 1950 did not exhibit the presence of any p element. Hence they are called empty strains. It has been found that there are two cycotypes of fruit flies. One is called a P cycotypes and the other an M cytotype. The difference is that p element is allowed to move in the M cycotyper, while the movement of the element is prevented in the P cycotype. Therefore, reciprocal crosses involving P and M cytotypes give different results.
It is to be noted here that the p element is inherited maternally in the flies and due to the transposition of the p elements the progeny flies face serious defects including sterility. This is known as hybrid dysgenesis.
TEs and mutation
Transposable elements may be called mutagens as they can induce mutation in various ways. If the transposon is inserted into a functional gene, it may damage the gene or alter the genic activity. After insertion presence of identical repeated sequences presents a problem in pairing during meiosis. LINES and SINES are found to be associated with some human diseases such as hemophilia A, Hemophilia B, SCID, porphyria, and Duchenne Muscular Dystrophy.
Some notable aspects of transposons
- Transposons may be regarded as selfish DNA because they tend to make their own copies.
- Transposons represent junk DNA because they are not of obvious benefit to their host.
- Because of the sequence similarities of all LINES and SINES, they form a large fraction of repetitive DNA in an organism.
- Retrotransposons are sometimes a threat to the survival of the host.
- Transposons sometimes add to the C value of an organism and therefore, they contribute to the C value paradox.
Human Genome Project
Human Genome Project (HGP) may be defined as an international research project with the aim of determining the sequence of base pairs that make up human DNA and of determining and locating approximately 20000-25000 genes of the human genome. HGP is considered a Megaproject to un-reveal approximately 3.3 billion base pairs.
HGP or Human Genome Project is an endeavour to characterize the whole human genome with the sequencing of DNA. However, the project has come into establishment from the urge to know the constitution of the human genome. With the discovery of improved techniques of molecular Biology such as cloning and DNA sequencing during the 1970S and early 1980S scientists began thinking of the possibility of sequencing a human genome containing 3 x 109 bp DNA present in 24 human chromosomes. This idea led to the launch of the Human Genome Project in 1990. Since sequencing of the human genome has a global impact, the project was launched jointly by a number of countries such as the United States of America, England, Japan, Germany, and France. However, in subsequent years many other countries joined hands in the project and a global consortium as Human Genome Organization (HUGO) was established to coordinate the efforts of the geneticists internationally.
History of HGP
Though HGP was launched in 1990, the first official funding for the project originated with the US DOE’s Office of Health and Environment Research in 1987. The project was planned for 15 years and it remained as one of the largest single investigative projects. In 1990, DOE and NIH in the United States came to an agreement to coordinate the plan and started running the project in 1990 with the goal to complete the work of the project in 15 years. The project was started under the leadership of Davis Galas director of the office of Biological and Environmental Research in the US DOE’s Office of science and James Watson in the NIH Genome Programme. Subsequently, Aristide Patrinos succeeded Galas and Francis Collins succeeded James Watson as the overall head of the project.
Because of widespread international cooperation and the advancement of genomic research, a rough draft of the genome could be prepared in 2000 and a complete one in 2003. Therefore, the goal of the project has been achieved two years before the target time. Although the main sequencing phase of HGP has been completed studies on DNA variation are in the run under International HapMap Project. The aim of this project is to identify single nucleotide polymorphism (SNP).
Objectives of HGP:
- To construct a detailed physical map of the entire human genome.
- To identify and locate all the human genes.
- To sequence the DNA of all 24 human chromosomes.
Progression of the Human Genome Project
1. Mapping of the human genome:
Progress in the mapping of the human genome was very rapid from the launching of the project in 1990. A complete physical map of the Y chromosome and chromosome 21 alongwith the RFLP map of the X and 22 autosomes was published in 1992. By 1996 a comprehensive and detailed STR map of the human genome was published and by 1997 a comprehensive map of 16354 discrete loci came to light. These discoveries helped researchers in cloning genes based on their location on the chromosomes. However, such mapping could only provide a low resolution (1-10 MB), and high-resolution mapping (-50 kb) can be achieved by radiation hybrid mapping, a modification of somatic cell hybridization mapping. The irradiation of human cells followed by their fusion with Chinese hamster cells in culture promoted the mapping of many human genes.
2. Sequencing human genome:
The complete sequence of the human genome could be achieved by 2003. However, the complete sequence of small chromosome 22 could be obtained first in 1999 after 9 years of initiation of the project. In the next year, the sequence of chromosome 21 was obtained. At the same time, the draft sequence of the whole human genome came to light. The information in the first drafts on human genome sequencing indicated that the human genome is more than 25 times the size of the genomes of Drosophila and Arabidopsis. The sequencing of the human genome has revealed that there are at least 25000-30000 genes in human chromosomes. On average per 145 kb of the human genome, there is one gene and on average one human gene is 2700 bp long. Each gene is composed of introns and exons and exons constitute 1.1% of the genome and the introns amount to about 24% of the genome. Whereas 75% of the genome appeared to be intergenic DNA. Now it has been revealed that there are only 22287 protein-coding genes, however, there also other are genes that specify RNA products such as rRNA, tRNA, snRNAs, and miRNAs.
Amount of data from HGP
The amount of data obtained in mapping and sequencing of the human genome if is stored in book form in which each page can record 1000 bp and if a book contains 1000 pages, then total genome data can only be accommodated in 3300 books. From this example, the amount of prolific sequencing data obtained from HGP can be realized. However, computer storage of this data can be loaded in a CD incorporating a volume of 786 megabytes.
Benefits from HGP
The use and interpretation of data obtained from HGP is still in the initial stage. The knowledge of the human genome may provide new awareness for medicines and biotechnology. Genetic tests now may determine one individual’s predisposition to a variety of ailments namely breast cancer, Alzheimer’s disease, haemostasic disorders, cystic fibrosis, liver diseases, Huntington’s disease, and many others. Simply DNA sequence data may help in a possibility of a person being affected by a serious genetic disease. The diseases of late onset may be detected in a person descended from a family which has a previous history of the disease.
By visiting the database of the human genome on the world wide web, a researcher in his investigation may narrow down his quest to any particular gene of interest. The evolutionary relationship of the specific human gene may also be established by comparing the gene sequence of humans with those of other organisms. A deeper understanding of the disease process at the level of molecular biology may also direct in determining new therapeutic procedures. In summary, the sequencing data from Human Genome Project may provide a new vista in the diagnosis of genetic diseases, new avenues to such disease treatment, and the determination of an evolutionary relationship between man and another animal.
DNA fingerprinting represents a molecular method of identification of an individual among many such individuals based on their similarity in DNA contents having variable number tandem repeats following DNA digestion with restriction endonuclease, electrophoretic separation of the DNA fragments, and Southern blotting.
Discovery of DNA Fingerprinting
DNA fingerprinting was discovered by English scientist Alec J. Jeffreys. He was the man who first reported the existence of VNTR in DNA and he pointed out that VNTR may be used for identification of a person or species.
Methods in Preparation of DNA Fingerprint
With a very little amount of DNA from a man, a DNA fingerprint may be prepared. Therefore, DNA isolated from a body cell may help to prepare a DNA fingerprint. If the amount of DNA is not sufficient, the same DNA may be increased by quantity by PCR method. However, the methods to prepare a fingerprint from the obtained DNA may be described as under:
1. The obtained genomic DNA from a cell is digested with the help of a restriction endonuclease. As a result, the DNA molecules are fragmented into pieces.
2. The DNA fragments are then separated by Gel electrophoresis. In this electrophoretic separation, the DNA fragments in the mixture are kept in the well at a side of a gel block of agarose which is then exposed to +ve and -ve electrodes in a trough containing buffer solution. The DNA fragments are negatively charged and therefore, they tend to migrate to the +ve electrode in the gel block. However, the smaller DNA fragments move faster towards the +ve electrode and the larger fragments trail behind. In this method, the large and small DNA fragments get separated by electrophoresis.
3. The separated DNA fragments are normally not visible in the gel block and to visualize the separated DNA fragments another technique is adopted. With the help of Southern blotting, the separated DNA fragments are taken on a nitrocellulose membrane.
4. Then the nitrocellulose membrane is exposed to radioactive probes which combine with the separated DNA fragments on the nitrocellulose filter/membrane.
5. Now over the nitrocellulose membrane one X-ray film is placed to record the location of the radioactive probes with the DNA fragments. In development, the X-ray film shows the separated DNA fragments in the form of bands. These black bands over the X-ray film represent the DNA fingerprint of a person.
Utility of DNA Fingerprinting
DNA fingerprinting is used to identify the real criminal. In many cases of crime, there may be several suspected persons. But to know and identify the real person involved in criminal activity, DNA fingerprinting may be a dependable tool. Murder and rape are two criminal activities of serious nature in society. The criminals usually leave the place after their activity is over and sometimes it becomes difficult to find out the real culprit. However, for the activity, many suspected persons may be identified, but the real murderer or rapist should be detected for punishment. In this exercise, DNA fingerprinting may be significantly helpful.
Usually at the crime site, the criminal leaves some of his belongings which may be a piece of hair, skin cells, drops of blood, or semen. Those may be taken into consideration for identifying the criminal. In case of criminal activity, the leftover organic part of the unknown criminal is taken to the laboratory and processed to obtain its DNA. The DNA obtained in this way is called evidence. The fingerprint from this evidence is prepared by the method described before. In the same way, the DNA fingerprints are prepared from all the suspected persons. The DNA profile of the evidence is compared with the same type of profile from the suspects. This comparison may help to identify very conclusively the real criminal from several suspected persons. Sometimes dispute arises regarding the paternity or maternity of a child. Such disputes may also be resolved by DNA fingerprinting. It is to be pointed out here that the DNA profile of the child matches 50% of the DNA profile of either of the parents, the father or the mother.