Analysis and Discussion of Available Genomic Tools for Obligate Inbreeding and Obligate Outcrossing Rhabiditic Species

Evolution of Genetics
During the nineteenth century may philosopherresearches forced their imaginations to conceptualize the variability in all organisms. Charles Darwin was the frontrunner and based on his extensive travels and studying all kinds of life forms, even fossils of plants and animals.

The Variation of Animals and Plants under Domestication The8 Variat ion of Animals and Plants under Domestication (1868) proposed a hypothetical mechanism of heredity as a provisional hypothesis called pangenesis. In the theory of evolution (pangenesis) of Hugo de Vries 1988 term for the same concept) he referred to heritability of character called the pangene whose transmission occurs from parent to offspring. The theory contained many facts but sadly without reasoning and contained explanations to phenomena such as atavisms, intermediate nature of hybrids, use and disuse of Lamarck, limb regeneration etc. He lacked imagination on a clear mechanism of inheritance. That he had the foresight to use the principle of inheritance to explain variability without any clues on meiosis, chromosomes or for that matter, much less genes, DNA, or the basic principles of Mendelian genetics such as random segregation and independent assortment is significant (to be discussed later).

Darwin even did simple crosses with snapdragons but based the results on Lamarcks theories. What is surprising is that Darwin, in his thinking years (1835-1870), paid absolutely no attention to the cell and developmental biology and genetics that was beginning to happen at the same time in Europe. Ref

Around the same time, in 1866 Mendel published results of his work from his classical work on pea plants. Mendel used peas based the knowledge that they were bisexual with both cross and self-pollinating mechanisms with many known traits and had short life span. He followed a scientific approach to make clear controlled cross pollinations to control the characters. He followed these very characters in the progenies to look for their inheritance and distributions and ensured that data was accurately collected and analyzed statistically, and finally presented data with inferences on the two laws of heredity to Versuche ber Pflanzen-Hybriden in the Verhandlungen des Naturforschenden Vereins zu Brnn, following two lectures he gave on the work in early 1865. The highlights of the work are there is a pair of contrasting (dominant and recessive alleles) characters which segregate independently and get assorted randomly but in certain definite proportions in progenies depending on whether they are dominant or recessive and which are predictable.

The term gene was coined by a Danish Botanist Wilhelm Johannsen in 1909 for these units of heredity. Genetics was named by William Bateson in 1905 for the study of the Science of Heredity.

A mention must be made that Mendel in his presentation acknowledged richly the contributions of his contemporaries as follows inexhaustible perseverance of Klreuter, Grtner, Herbert, Lecoq, Wichura and others, Mendel further observed that .so far, no generally applicable law governing the formation and development of hybrids has been successfully formulated.

He also exclaimed that none of these researchers made any effort to enquire on determining the number of different groups to which different progenies can belong and also to classify them according to different generations as also on the statistical inferences. He wrote, none has been carried out to such an extent and in such a way as to make it possible to determine the number of different forms under which the offspring of the hybrids appear, or to arrange these forms with certainty according to their separate  generations, or definitely to ascertain their statistical relations.

The true significance of Mendels work had to wait till his paper  was rediscovered by De Vries, Correns and Tschermak 40 years after its publication.

Ever since the principles of inheritance and variation were re-established Genetics, coined by Bateson has become the mainstay for more experimental approaches to creating variability and ensuring heritability of desired characters from parents to offspring in crop improvements. The principles of inheritance were originally analyzed and inferred from painstaking but clear cross pollinations in pea plants for many years. The Mendelian principles proposed that discrete units of characters, factors (now called genes), were inherited by offspring from parents. They are assorted and segregated independently. Mendel discovered all this without any idea of Genes genes, Meiosis meiosis whose roles in heredity were elaborated years after his death and which showed how Mendels laws are carried out. This stimulated considerable researches on verifying these principles.  Drosophila melanogaster Drosophila melanogaster attracted maximum attention for applications of Mendelian genetics. Morgan established the Mendelian-Chromosome theory of Heredity.

The Gene was thus born. It is not clear when exactly the term gene was coined but thie word was used in the literature. But (website o the The National Human Genome Research Institute, NIH, USA) describes that in 1869, in a hospital Johann Friedrich Miescher isolated a substance called nuclein from a pus of a wound. Later this substance came to be known as nucleic acid. In 1879 Walter Flemming observed mitosis in salamander embryos developed stain to stain chromosomes clearly and described chromosome behavior during mitotic division.

By 1900 Mendels concepts (factors) were getting nearly understood when three European Botanists - Carl Correns, Hugo DeVries, and Erich von Tschermak, unaware of each other, independently reviewed the literature before publishing their own respective results on the laws of inheritance from their experiments of pea plants (1902). In 1905 a Mendels supporter, William Bateson felt the need for a new word genetics is supposed to have suggested so in letter before W. Johanssen coined the word gene as well as phenotype and genotype (1909 cited in ..  Roberts, H. F. Plant Hybridization before Mendel. Princeton Princeton University Press, 1929 and later It is significant to mention that in 1902 itsel the Alkaptoneuria disease also was observed to inherited as per Mendels laws Garod, W, cited in This year (1902) Suttons observations from grasshopper cells confirmed Theodor Boveris earlier observations (during 1880s and 1890s) that  chromosome numbers are reduced to half the original numbers as egg cells mature and also that  the segregation pattern of chromosomes during meiosis matched the segregation patterns of Mendels genes.    This also important support to the chromosomal theory of heredity and further support was obtained from .experimental observations of Thomas Hunt Morgan, Alfred Sturtevant, Hermann Muller and Calvin Bridges that genes are arranged in a linear waay on chromosomes and that they are linked. In 1911 in fruitflies.

In 1941 working on x-irradiated Neurospora crassa Beadle and Tatum found that genes mutate and cause biochemical deficiencies making it imperative for the organism to to grow on an artificial medium only when supplemented with a particular nutrient, an amino acid in their example. Their proposal further clarified the understanding of the gene as a functional unit which governs the formation of only one enzyme.

Earlier in 1928 Griffith, working on  two strains of Streptococcus pneumonia,  harmless (R) and virulent (S) observed that the R strain could get converted into harmful deadly S strain by simply injecting simultaneously the R strain as well as the heat killed S strain into mice. The mice though injected with killed S strain was not unexpected to develop the pneumonia disease but they did. Such results were observed for a long time in Oswald Averys lab till 1940s. In 1941 Avery along with
A gene as broadly known today is a unit of heredity in a living or dead organism with a predetermined function functioning in a synchronous manner in synchrony with millions of other genes in the total primary genetic configuration of an organism. It is a strand of a unique DNA structure with a code for ordering production of a protein and RNA for a specific function. This crisp short description of a gene has been facilitated by many scientists working on different directions simultaneously and sequentially. Every organism has millions of genes arranged in a linear fashion in the whole complement called the genome.

The understanding of gene was a concept in the Classical Era described so far. A gene as broadly known today is a unit of heredity in a living or dead organism with a predetermined functioning in a synchronous manner in synchrony with millions of other genes in the total primary genetic configuration of an organism. It is a strand of a unique DNA structure with a code for ordering production of a protein and RNA for a specific function. This crisp short description of a gene has been facilitated by many scientists working on different directions simultaneously and sequentially. Every organism has millions of genes arranged in a linear fashion in the whole complement called the genome.

The broad area of Genetics has proliferated quantitatively and ramified qualitatively into many other disciplines. The total evolution of Genetics may be considered in different eras.

In the Classical described thus far the attempts have repeatedly been made to dexcribe gene as a structural and a functional unit with no references to possibilities of architectural diversities. In the following discussions an idea of how different this concept can be is discussed.

Watson and Crick
J. D. Watson was a molecular biologist and Framcis Crick was a molecular biologist, physicist and a neuroscientist, are the codiscoverers of the DNA structure working the the Cavendish Laboratory,   . The first announcement of the DNA structure was made by the Director of the Cavendish Laboratory, Sir Lawrence Bragg at Solway Conference on proteins in Belgium in 1953. Watson and Crick published this paper in Nature 1953.

They acknowledge Pauling and Coreys paper made available to them prior to its publication and found some objections to the observations made by them. They also had on hand a paper in press by Fraser. Again Watson and cricks convictions about their own model was strong enough to add their comments in their paper in Nature (1953).

According to Watson and Crick the DNA has two helical chains each coiled around the same axis. Based on normal chemical assumptions the two chains but not their bases are related by a dyad perpendicular to the fibre axis. Both chains follow right handed helices but due to the dyad, the sequences of the atoms in the two chains run in opposite directions.

They were fully aware of the then available information from other active researchers on nucleic acids (from accessexcelllence)
DNA is made up of subunits which scientists called nucleotides.
Each nucleotide is made up of a sugar, a phosphate and a base.
There are 4 different bases in a DNA molecule
Adenine (a purine)
Cytosine (a pyrimidine)
Guanine (a purine)
Thymine (a pyrimidine)
The number of purine bases equals the number of pyrimidine bases
The number of adenine bases equals the number of thymine bases
The number of guanine bases equals the number of cytosine bases

The basic structure of the DNA molecule is helical, with the bases being stacked on top of each other

They both made good use of their expertise as well some additional information obtained informally. It was primarily based on the x-ray crystallography data from Rosalynd Franklin and Maurice Wilkins.

Working with nucleotide models made of wire, Watson and Crick attempted to put together the puzzle of DNA structure in such a way that their model would account for the variety of facts that they knew described the molecule. Once satisfied with their model, they published their hypothesis, entitled Molecular Structure of Nucleic Acids A Structure for Deoxyribose Nucleic Acid in the British journal Nature (April 25, 1953. volume 171737-738.) It is interesting to note that this paper has been cited over 800 times since its first appearance

Kary Banks Mullis and Polymerase Chain Reaction
Kary Banks Mullis is a self-motivating achiever biochemist with broad and diverse and even unrelated interests but with a strong sense of adventure and attitude of perseverance to pursue the convictions of imaginations till achieved. He is a well known popularly as intellectual maverick and a Chemistry Nobel Laureate for his contributions to the development of Polymerase Chain Reaction (PCR) in1993. In fact the idea for this most popular technology was originally available in a paper by Kleppes et al (1971). Kleppes et al.(1971) described an in vitro enzymatic method to replicate a short DNA template with primers.

But the significance of this idea was best recognized only by Kary Mullis in 1986.  While working on the idea of using DNA polymerase to copy a desired DNA sequence by using two primers to bracket it. In his imagination this technique was expected to facilitate copying small strand of DNA almost an infinite number of times.

One of the complications of this was that high heat used at the beginning of the experiment destroyed the DNA polymerase. Logically he turned his attention to the DNA polymerase enzyme from Thermophilus aquaticus Thermus aquaticus. Taq polymerase  is a thermostable  DNA polymerase DNA polymerase named after Thermophilic thermophilic bacterium  Thermus aquaticus Thermus aquaticus which it was originally isolated by Thomas D. Brock Thomas D. Brock in 1965.

T. aquaticus is a  Bacterium bacterium found in the Hot springs hot springs and Hydrothermal vent hydrothermal vents, and Taq polymerase was identified as an  Enzyme enzyme able to withstand the protein-denaturing conditions (high temperature, temperature optimum 80C) required during PCR. DNA polymerase was first isolated from Thermus aquaticus in 1976. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus by A Chien, D B Edgar, and J M Trela in Journal of Bacteriology 127 (3) 15501557.) The first advantage that was found for this thermostable (temperature optimum 80C) DNA polymerase was that it could be isolated in a purer form (free of other enzyme contaminants) than could the DNA polymerase from other sources. Subsequently T. aquaticus was the source of many other enzymes such as Taq I restriction enzyme, aldolase, dehydrogenases, alkaline phosphatases RNA polymerases, DNA ligase DNA ligase,  Alkaline phosphatase Alkaline Phosphatase,  Oxidase NADH Oxidase,  Isocitrate dehydrogenase Isocitrate Dehydrogenase,  Maltase Amylomaltase, and the ever-popular  Lactate dehydrogenase Fructose 1,6-Bisphosphate-Dependent L-Lactate Dehydrogenase  all of which have properties of tolerating biologically inactivating high temperatures. Such researches also stimulated great interest in looking for enzymes in many other thermophilic organisms. One such organism is Pyrococcus furiosus Pyrococcus furiosus whose polymerase called Pfu was compared with Taq DNA polymerase and found that Pfus superior thermostability and proofreading properties compared to thermostable Taq polymerases as well as those of other organisms. Similarly Pfus thermostability and proofreading properties were superior compared to other thermostable polymerases.. Pfu DNA polymerase possesses 3 to 5  Exonuclease exonuclease proofreading activity, meaning that it works its way along the DNA from the  5 end 5 end to 3 end 3 end and corrects Nucleotide nucleotide-misincorporation errors. This implies that PCR fragments  generated by Pfu DNA polymerase is likely to show fewer errors than those from Taq-polymerase inserts. Consequently Pfu is more commonly used for molecular cloning of PCR fragments than the historically popular Taq.

Commercially available Pfu typically results in an error rate of 1 in 1.3 million base pairs and can yield 2.6 mutated products when amplifying 1kb fragments using PCR. However, Pfu is slower and typically requires 12 minutes per cycle to amplify 1kb of DNA at 72 C. Using Pfu DNA polymerase in PCR reactions also results in blunt-ended PCR products.

Pfu DNA polymerase is hence superior for techniques that require high-fidelity DNA synthesis, but can also be used in conjunction with Taq polymerase to obtain the fidelity of Pfu with the speed of Taq polymerase activity

Polymerase Chain Reaction
Polymearse Chain Reaction - Technique
This reaction has become well integrated into biology and medicine investigations. Most of the applications of this are in amplifying (multiplying) a single or a few copies of DNA (gene) exponentially resulting in very large numbers of copies (thousands to millions of copies) of a particular DNA sequence. The basis of this method is a repeated cyclic process of three differential temperature treatments (heating and cooling) to a reaction mixture containing DNA fragment to be amplified. The different temperatures (heating and cooling) are meant for DNA melting and DNA replication respectively with a DNA polymerase.  Primer (molecular biology) Primers (short DNA fragments) containing sequences complementary to the target region along with a Taq polymerase DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion Chain reaction chain reaction in which the DNA template is Exponential growth exponentially amplified. PCR can be extensively modified to perform a wide array of Genetic engineering genetic manipulations.

The cycling reactions
There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time.

Denaturation at 94C
1 minute 94 C During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example  the extension from a previous cycle).

Annealing at 54C 45 seconds, forward and reverse primers
The primers are jiggling around, caused by the Brownian motion. Ionic bonds are constantly formed and broken between the single stranded primer and the single stranded template. The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. Once there are a few bases built in, the ionic bond is so strong between the template and the primer, that it does not break anymore.
extension at 72C 2 minutes, only dNTPs

This is the ideal working temperature for the polymerase. The primers, where there are a few bases built in, already have a stronger ionic attraction to the template than the forces breaking these attractions. Primers that are on positions with no exact match, get loose again (because of the higher temperature) and dont give an extension of the fragment. The bases (complementary to the template) are coupled to the primer on the 3 side (the polymerase adds dNTPs from 5 to 3, reading the template from 3 to 5 side, bases are added complementary to the template)

The ladder is a mixture of fragments with known size to compare with the PCR fragments. Notice that the distance between the different fragments of the ladder is logarithmic. Lane 1  PCR fragment is approximately 1850 bases long. Lane 2 and 4  the fragments are approximately 800 bases long. Lane 3  no product is formed, so the PCR failed. Lane 5 multiple bands are formed because one of the primers fits on different places.

Because both strands are copied during PCR, there is an exponential increase of the number of copies of the gene. Suppose there is only one copy of the wanted gene before the cycling starts, after one cycle, there will be 2 copies, after two cycles, there will be 4 copies, three cycles will result in 8 copies and so on. After 35th cycle there would be 68 billion (236) copies from one single fragment.

To check whether the PCR generated the anticipated The amplified DNA fragment (o Amplicon amplicon) generated by PCR, the products are checked for trueness to the expectations by agarose gel electrophoresis along with standard marker DNA fragments of known molecular weights. This method provides sizes of the test molecules when compared with the bands of DNA ladder formed

Optimizing PCR
Due to both established variations of this technique and newly developing broad applicability opportunities of this method developed by Kary Mullis, PCR is indispensible for a majority of basic and applied biological investigations. In many situations the technique has to be subjectively standardized before real applications on a case to case basis. As mentioned earlier, although most extensively used, the method has some limitations such as misreading of sequences, its sensitivity to contaminating DNA and erroneous DNA products and others. A number of techniques and procedures have been developed for optimizing PCR conditions  (PCR from problematic templates. Focus 221 p.10 (2000). Helpful tips for PCR. Focus 221 p.12 (2000). Separate prerun of agarose gel without the PCR samples is run as a standard procedure. PCR  set up is maintained in a separate place thoroughly cleaned before and after the electrophoresis runs. Primer design is extremely important for getting good and reliable results.

Since then there have been major innovations made, as applications of PCR have been essential to the progression of certain areas in science. These include the mapping of the human genome project, single sperm analysis, molecular archaeology and ancient DNA, molecular ecology and behaviour, disease diagnosis and drug discovery.

PCR has enabled the analysis of molecules from the past in order to examine species that may now be extinct and to determine their relationships to present day organisms. One example of this analysis is, DNA from a 5000 year old frozen mummy, the Tyrolean ice man, was analysed in a hypervariable region of mitochondrial genome and this late Neolithic individuals mitochondrial type was found in 13 of 1200 people and was closely related to the types determined from central and northern Europe populations.

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Classification of organisms
Molecular archaeology
Mutation detection
Cancer research
Detection of pathogens
DNA fingerprinting
Drug discovery
Genetic matching
Genetic engineering
Pre-natal diagnosis
Mutation screening
 Drug discovery
 Classification of organisms
 Molecular Archaeology
 Molecular Epidemiology
 Molecular Ecology
 Genomic cloning
 Site-directed mutagenesis
 Gene expression studies

Applications of PCR
Basic Research

Mutation screening
 Drug discovery
 Classification of organisms
 Molecular Archaeology
 Molecular Epidemiology
 Molecular Ecology
 Genomic cloning
 Site-directed mutagenesis
 Gene expression studies

Applied Research
Genetic matching
 Detection of pathogens
 Pre-natal diagnosis
 DNA fingerprinting
 Gene therapy

Applications of PCR

Molecular Identification
Molecular Archaeology
Molecular Epidemiology
Molecular Ecology
DNA fingerprinting
Classification of organisms
Pre-natal diagnosis
Mutation screening
Drug discovery
Genetic matching
Detection of pathogens
Genomic cloning
Human Genome Project

Genetic Engineering
Site-directed mutagenesis
Gene expression studies

Variations of PCR technique from google

Allele specific PCR This is based on single nucleotide polymorphisms requiring exact prior information on the DNA sequence including allele differences and used both for diagnostics and cloning. But under stringent conditions this less efficient.

Polymerase cycling assembly Assembly PCR or Polymerase Cycling Assembly (PCA) PCR is performed on a pool of long oligonucleotides with short overlapping sequences for artificial synthesis of long synthesis of long oligonucleotde sequences.

Assymmetric PCR It is used in hybridization and sequencing. This amplifies one DNA strand in a double stranded DNA template. One of the two primers needed in excess for the strand directed sequence. This has been further modified to accelerate the slow rate of reaction.

Helicase-dependent amplification Helicase-dependent amplification is the same as the normal  PCR, but uses a constant temperature rather than cycling through denaturation and annealingextension cycles.  DNA helicase DNA helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation. Hot-start PCR (page does not exist) Hot-start PCR a technique that reduces non-specific amplification during the initial set up stages of the PCR. It may be performed manually by heating the reaction components to the melting temperature before adding the polymerase. Specialized enzyme systems have been developed that inhibit the polymerases activity at ambient temperature, either by the binding of an Antibody antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. Hot-startcold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.

Main article  Variants of PCR
Allele-specific PCR (page does not exist) Allele-specific PCR a diagnostic or cloning Single-nucleotide polymorphism single-nucleotide polymorphisms (SNPs) (single-base differences in DNA). It requires prior knowledge of a DNA sequence, including differences between Allele alleles, and uses primers whose 3 ends encompass the SNP. PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence. See SNP genotyping SNP genotyping for more information.

Polymerase cycling assembly Assembly PCR or Polymerase Cycling Assembly (PCA) artificial synthesis of long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments, thereby selectively producing the final long DNA product.

Asymmetric PCR (page does not exist) Asymmetric PCR preferentially amplifies one DNA strand in a double-stranded DNA template. It is used in Sequencing sequencing and hybridization probing where amplification of only one of the two complementary strands is required. PCR is carried out as usual, but with a great excess of the primer for the strand targeted for amplification. Because of the slow amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required.

A recent modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.

Helicase-dependent amplification Helicase-dependent amplification similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealingextension cycles.

DNA helicase DNA helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.

Hot-start PCR (page does not exist) Hot-start PCR a technique that reduces non-specific amplification during the initial set up stages of the PCR. It may be performed manually by heating the reaction components to the melting temperature (e.g., 95C) before adding the polymerase. Specialized enzyme systems have been developed that inhibit the polymerases activity at ambient temperature, either by the binding of an  Antibody antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. Hot-startcold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.

Intersequence-specific PCR (page does not exist) Intersequence-specific PCR (ISSR) a PCR method for DNA fingerprinting that amplifies regions between simple sequence repeats to produce a unique fingerprint of amplified fragment lengths. Inverse polymerase chain reaction Inverse PCR is commonly used to identify the flanking sequences around Genomic genomic inserts. It involves a series of  Restriction digest DNA digestions and  Self ligation self ligation, resulting in known sequences at either end of the unknown sequence. Ligation-mediated PCR (page does not exist) Ligation-mediated PCR uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers it has been used for DNA sequencing DNA sequencing,  Genome walking genome walking, and  DNA footprinting DNA footprinting.

Methylation-specific PCR Methylation-specific PCR (MSP) developed by Stephen Baylin and Jim Herman at the Johns Hopkins School of Medicine, and is used to detect methylation of CpG islands in genomic DNA. DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCRs are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences. At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation.

Miniprimer PCR (page does not exist) Miniprimer PCR uses a thermostable polymerase (S-Tbr) that can extend from short primers (smalligos) as short as 9 or 10 nucleotides. This method permits PCR targeting to smaller primer binding regions, and is used to amplify conserved DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene.  Multiplex Ligation-dependent Probe Amplification Multiplex Ligation-dependent Probe Amplification (MLPA) permits multiple targets to be amplified with only a single primer pair, thus avoiding the resolution limitations of multiplex PCR (see below).

Multiplex-PCR Multiplex-PCR consists of multiple primer sets within a single PCR mixture to produce Amplicon amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes, i.e., their base pair length, should be different enough to form distinct bands when visualized by Gel electrophoresis gel electrophoresis.

Nested PCR Nested PCR increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3 of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.
Overlap-extension PCR Overlap-extension PCR a Genetic engineering genetic engineering technique allowing the construction of a DNA sequence with an alteration inserted beyond the limit of the longest practical primer length.

Q-PCR Quantitative PCR (Q-PCR) used to measure the quantity of a PCR product (commonly in real-time). It quantitatively measures starting amounts of DNA, cDNA or RNA. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. Quantitative real-time PCR has a very high degree of precision. QRT-PCR methods use fluorescent dyes, such as Sybr Green, EvaGreen or  Fluorophore fluorophore-containing DNA probes, such as TaqMan TaqMan, to measure the amount of amplified product in real time. It is also sometimes abbreviated to Real-time PCR RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions, since RT-PCR commonly refers to  RT-PCR reverse transcription PCR (see below), often used in conjunction with Q-PCR.

Reverse Transcription PCR (RT-PCR RT-PCR) for amplifying DNA from RNA.  Reverse transcriptase Reverse transcriptase reverse transcribes  RNA RNA into  CDNA cDNA, which is then amplified by PCR. RT-PCR is widely used in  Expression profiling expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites. If the genomic DNA sequence of a gene is known, RT-PCR can be used to map the location of  Exons exons and  Introns introns in the gene. The 5 end of a gene (corresponding to the transcription start site) is typically identified by  RACE (biology) RACE-PCR (Rapid Amplification of cDNA Ends).

Solid Phase PCR (page does not exist) Solid Phase PCR encompasses multiple meanings, including Polony Polony Amplification (where PCR colonies are derived in a gel matrix, for example), Bridge PCR (primers are covalently linked to a solid-support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR (where conventional Solid Phase PCR can be improved by employing high Tm and nested solid support primer with optional application of a thermal step to favour solid support priming).

Thermal asymmetric interlaced PCR TAIL-PCR (page does not exist) TAIL-PCR) for isolation of an unknown sequence flanking a known sequence. Within the known sequence, TAIL-PCR uses a nested pair of primers with differing annealing temperatures a degenerate primer is used to amplify in the other direction from the unknown sequence.

(Step-down PCR) a variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3-5C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3-5C) below the primer Tm. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles. PAN-AC (page does not exist) PAN-AC uses isothermal conditions for amplification, and may be used in living cells. Universal Fast Walking (page does not exist) Universal Fast Walking for genome walking and genetic fingerprinting using a more specific two-sided PCR than conventional one-sided approaches (using only one gene-specific primer and one general primer - which can lead to artefactual noise) by virtue of a mechanism involving lariat structure formation. Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nested PCR for rapid amplification of genomic DNA ends).

b. Model Systemsi. What is a good model See Wikipedia
Even with increasing number of researchers and facilities it may jus t not be possible to study even one organism in fullest depth. A model system id required for a situation when there are two basic questions of enquiry  the one best method for control and or prevention of any one human disease the other being does the present information provide every kind of information required to understand all its life mechanisms    

Concerning human biology and pathology in general there is always the issue which organism can provide the nearly closest answers to any of the human problems. Even assuming there could be one hypothetical organism whose biology is known and understood to the fullest extent   maximum there is no guarantee that this organisms could be used to understand and develop mechanisms for control and prevention of a problem in any other organism including human beings.

Yet the need for an organism is warranted which can be extensively studied intensively and extensively. In the course of accumulating information one after the other, many organisms have been used for unraveling a mechanism or development of testable theory based experiments and so on. For example whatever is known today about the genetics and molecular genetics is based a number of organisms.

For example Mendel performed his experiments with pea plants and discovered the famous two foundation laws of inheritance. In the paper read at ..  out of the total of 15,226 word, he devoted 706 words for a section  under the heading SELECTION OF THE EXPERIMENTAL PLANTS. In this section Mendel reveals his sense of accuracy in describing how and why he chose the pea plants. He wrote The value and utility of any experiment are determined by the fitness of the material to the purpose for which it is used, and thus in the case before us it cannot be immaterial what plants are subjected to experiment and in what manner such experiment is conducted. The experimental plants must necessarily

1. Possess constant differentiating characteristics. 2. .. In all, thirtyfour more or less distinct varieties of Peas were obtained from several seedsmen and subjected to a two years trial. Later he justified how the plants chosen were indeed fitting into his original enquiries on the regularity of the recurrence of the characters of the hybrid forms and their consistent proportions.

In the course of development of biology many organisms were chosen based on the need and basis of simple enquiry. In the process many organisms got selected more frequently because more and more of information got collected. This became the norm for the frequent choice of an organism for an experiment. But more recently the trend has changed so drastically that to dissect out an answer to a problem in one organism another related organisms has to be chosen. This is on the assumption that the solutions or leads obtained from the related organism can be extrapolated and extended to the test organism itself. For example any human related problem another mamma such as mouse or a monkey was the popular choice.    

After all these, now a clear concept on what a model organism should be, has emerged. Essentially a model organism has to be a non-human organism which has been extensively studied  for understanding a biological process in anticipation  that any findings with the model organisms would become applicable to other organisms. The situation has greater relevance to understanding human diseases. The model organism for investigation on causes and treatments for human disease so that the the findings may get applied to human situations. The need for a model organisms in such situations is also imperative on ethical grounds. This logic is now based on the consensus that all the organisms have a common ancestry. This is also due to the understanding that there has been evolutionary conservation in the genetic material and therefore the conservation of metabolic and developmental pathways. The only precaution that needs to be taken care of is that generalizations and simple extrapolations should not be generalized.  
Some Basic Quantifiable Criteria Needed in Model System
The organism to used should be easy to rear andor culture.
Its rearingoperation size should be convenient
It is economic and inexpensive to operate.
The organism for use should have a short life cycle

The organism must have an easy reproductive mechanism with very high rate of multiplication  
The organism to be used should have the ability to be grown in culture on well defined  simple well established media for genetic manipulations

It should have fitness into the experimental plan for anticipated results to have high potential for  providing economically important results in consequent experiments
The organism should have a wide audience of interested researchers and public as well.

There must be high level of positive interactions between and among these criteria.

Choice of a Model Organism
Generally the choice agree with most of the above parameters but may not agree with all. Over and above the above factors, the choice of an organism is made or becomes very stringent due to many reasons. The organism chosen based on the above parameters may satisfy all the above parameters but is not suitable because of lack of parallel comparisons with the test organism. The choices may have to be made on other considerations such as emphasis on using genetic models such as fruit fly or nematode (both with very rich genetic information, and high rates of multiplication in short life cycles), experimental models and or genomic models because of their key positions in the evolutionary tree. Historically and as mentioned earlier there are just a few including the NIH model organisms (NIH Model Organisms) which are characterized extensively genetically and in the sense of molecular biology.        

Choices of organism also include the negative traits that the organism may have. One organism may agree with all parameters but may have some technical limitations and difficulties such as large amounts of junk DNA. Organism for potential good use may have to be disregarded on other considerations such as ethical andor environmental considerations.

On the whole the many model organisms that have been developed and used may belong t any of the major life forms, from viruses to prokaryotes and eukaryotes including protists, fungi, plants, invertebrates and vertebrates.        

Organisms may have to be based on specific research objectives which makes the choice more difficult. They are Sexual selection and sexual conflict,  Hybrid zones, and ecological genomics, and ecological zones

Neurosota crassa
E coli


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