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After the DNA is run on the gel, the polymorphic bands or the bands cosegregating with a target trait are tagged. The bands identification is either done by calculating their relative mobilities (Rm / Rf values in case of isoenzymes) or by calculating their molecular weights (in case of proteins and DNA).

There are several methods to calculate molecular weights of DNA bands on the gel. Graphical approach is one of them. The approaches include (1) use of computer software to generate molecular weights and (2) running unknown DNA fragments with the fragments of known molecular weights (standard markers available from private companies, see examples in the figure). An example is given here (see figure) to illustrate the method to calculate molecular weights of unknown DNA fragments. This method is usually called as Graphical Approach.

In genetics, microRNAs (miRNA) are single-stranded RNA molecules of 21-23 nucleotides in length, which regulate gene expression. miRNAs are encoded by genes from whose DNA they are transcribed but miRNAs are not translated into protein (i.e. they are non-coding RNAs); instead each primary transcript (a pri-miRNA) is processed into a short stem-loop structure called a pre-miRNA and finally into a functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to down-regulate gene expression. They were first described in 1993 by Lee and colleagues in the Victor Ambros lab .

MicroRNAs can be encoded by independent genes, but also be processed (via the enzyme Dicer) from a variety of different RNA species, including introns, 3' UTRs of mRNAs, long noncoding RNAs, snoRNAs and transposons.

The genes encoding miRNAs are much longer than the processed mature miRNA molecule; miRNAs are first transcribed as primary transcripts or pri- miRNA with a cap and poly-A tail and processed to short, 70-nucleotide stem-loop structures known as pre-miRNA in the cell nucleus. These pre- miRNAs are then processed to mature miRNAs in the cytoplasm by interaction with the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC). This complex is responsible for the gene silencing observed due to miRNA expression and RNA interference. The pathway in plants varies slightly due to their lack of Drosha homologs; instead, Dicer homologs alone effect several processing steps. The pathway is also different for miRNAs derived from intronic stem-loops; these are processed by Dicer but not by Drosha. Either the sense strand or antisense strand of DNA can function as templates to give rise to miRNA.

In cell biology, a mitochondrion is a membrane-enclosed organelle found in most eukaryotic cells These organelles range from 0.5–10 micrometers (µm) in diameter. Mitochondria are sometimes described as “cellular power plants” because they generate most of the cell’s supply energy in form of adenosine triphosphate (ATP), used as a source of chemical energy. In addition to supplying cellular energy, mitochondria are involved in a range of other processes, such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth. Mitochondria have been implicated in several human diseases, including mitochondrial disorders and cardiac dysfunction, and may play a role in the aging process.

Several characteristics make mitochondria unique. The number of mitochondria in a cell varies widely by organism and tissue type. Many cells have only a single mitochondrion, whereas others can contain several thousand mitochondria. The organelle is composed of compartments that carry out specialized functions. These compartments or regions include the outer membrane, the intermembrane space, the inner membrane, and the cristae and matrix. Mitochondrial proteins vary depending on the tissue and the species. In humans, 615 distinct types of proteins have been identified from cardiac mitochondria; whereas in Murinae (rats), 940 proteins encoded by distinct genes have been reported. The number of proteins encoded in plant mitochondrial DNA is probably not much higher than the number encoded in mammalian mitochondria.Experiments have shown that most of the protein synthesis in isolated maize mitochondria can be accounted for by some 18-20 polypeptidesThe mitochondrial proteome is thought to be dynamically regulated. Although most of a cell’s DNA is contained in the cell nucleus, the mitochondrion has its own independent genome. Further, its DNA shows substantial similarity to bacterial genomes.

The biological diversity of each country is a valuable and vulnerable natural resource. Sampling, identifying, and studying biological specimens are among the first steps toward protecting and benefiting from biodiversity. Knowledge about biodiversity and the ability to identify organisms that comes with it are global public goods. That is, this knowledge can create benefits for all countries - benefits that no one country can create by itself.

DNA Barcoding: Documenting Biodiversity with a Gene Sequence

“DNA barcoding” is an exciting new tool for taxonomic research. The DNA barcode is a very short, standardized DNA sequence in a well-known gene. It provides a way to identify the species to which a plant, animal or fungus belongs. The Consortium for the Barcode of Life (CBOL) is promoting international partnerships that will enable people in all countries to better understand and protect their biodiversity.

DNA barcoding is a taxonomic method that uses a short genetic marker in an organism’s mitochondrial DNA to identify it as belonging to a particular species. It is based on a relatively simple concept: most eukaryote cells contain mitochondria and mitochondrial DNA (mtDNA) has a relatively fast mutation rate, which results in significant variance in mtDNA sequences between species and, in principle, a comparatively small variance within species.

What is FISH ?

Fluorescent in situ hybridization is a technique in which single-stranded nucleic acids (usually DNA, but RNA may also be used) are permitted to interact so that complexes, or hybrids, are formed by molecules with sufficiently similar, complementary sequences. Through nucleic acid hybridization, the degree of sequence identity can be determined, and specific sequences can be detected and located on a given chromosome. Fluorescent in situ hybridization (FISH) is a powerful technique for detecting RNA or DNA sequences in cells, tissues, and tumors. FISH provides a unique link among the studies of cell biology, cytogenetics, and molecular genetics.

Principle of FISH

FISH involves the preparation of short sequences of single-stranded DNA, called probes, which are complementary to the DNA sequences the researchers wish to paint and examine. These probes hybridize, or bind, to the complementary DNA and, because they are labeled with fluorescent tags, allow researchers to see the location of those sequences of DNA. Unlike most other techniques used to study chromosomes, which require that the cells be actively dividing, FISH can also be performed on non dividing cells, making it a highly versatile procedure.

The transfer foreign gene to the plant cell is a very crucial method to production of transgenic plant. So improvement of plant quality requires to transfer our interest gene or we can say useful for production of our interest product. To achieve genetic transformation in plants, we need the construction of a vector (genetic vehicle) which transports the genes of interest, flanked by the necessary controlling sequences i.e. promoter and terminator, and deliver the genes into the host plant. The two kinds of gene transfer methods in plants are.

1. Vector-mediated or Indirect Gene Transfer

Among the various vectors used in plant transformation, the Ti plasmid of Agrobacterium tumefaciens has been widely used. This bacteria is known as “natural genetic engineer” of plants because these bacteria have natural ability to transfer T-DNA of their plasmids into plant genome upon infection of cells at the wound site and cause an unorganized growth of a cell mass known as crown gall. Ti plasmids are used as gene vectors for delivering useful foreign genes into target plant cells and tissues. The foreign gene is cloned in the T-DNA region of Ti-plasmid in place of unwanted sequences.

To transform plants, leaf discs (in case of dicots) or embryogenic callus (in case of monocots) are collected and infected with Agrobacterium carrying recombinant disarmed Ti-plasmid vector. The infected tissue is then cultured (co-cultivation) on shoot regeneration medium for 2-3 days during which time the transfer of T-DNA along with foreign genes takes place. After this, the transformed tissues (leaf discs/calli) are transferred onto selection cum plant regeneration medium supplemented with usually lethal concentration of an antibiotic to selectively eliminate non-transformed tissues. After 3-5 weeks, the regenerated shoots (from leaf discs) are transferred to root-inducing medium, and after another 3-4 weeks, complete plants are transferred to soil following the hardening (acclimatization) of regenerated plants. The molecular techniques like PCR and southern hybridization are used to detect the presence of foreign genes in the transgenic plants.

DNA Sequencing

DNA sequencing is the determination of the precise sequence of nucleotides in a sample of DNA. Sequencing can be done on a short fragment of DNA to cover the entire genome. The maps thus constructed using the nucleotide sequences in the correct order is called as sequence map.

In 1977, twenty-four years after the discovery of the structure of DNA, two separate methods for sequencing DNA were developed: the chain termination method (Sanger et al., 1977) and the chemical degradation method (Maxam and Gilbert, 1977). Both methods were equally popular to begin with, but, for many reasons, the chain termination method is the method more commonly used today. In chemical degradation method, chemicals specific to each base remove the last base, which has been labelled. The method uses toxic chemicals which required high care while handling.

In chain termination method, DNA polymerase extends a DNA strand in which dideoxynucleotide becomes incorporated terminating the chain extension (phosphodiester formation) at the 3’OH site making an array of fragments differing in length. This method is based on the principle that single-stranded DNA molecules that differ in length by just a single nucleotide can be separated from one another using polyacrylamide gel electrophoresis.

Eastern blotting is a technique to detect protein post translational modifications (PTM) and is an extension of the biochemical technique of western blotting. There are multiple versions of the method, but in general proteins or lipids are blotted from one or two-dimensional SDS-PAGE gel on to a PVDF or nitrocellulose membrane are analyzed for post-translational protein modifications using probes that may detect lipids, carbohydrate, phosphorylation or any other protein modification. Eastern blotting should be used to refer to methods that detect their targets through specific interaction of the PTM and the probe, distinguishing them from a standard Far-western blot. The most common application is lectin blotting, however, a variety of other probes can be used for detecting different PTMs.

History and Multiple Definitions

Definition of the term Eastern blotting is somewhat confused due to multiple sets of authors dubbing a new method as Eastern blotting, or a derivative therof. All of the definitions are a derivative of the technique of Western blotting developed by Towbin in 1979. The current definitions are summarized below in order of the first use of the name, however all are based on some earlier works. In some cases, the technique had been in practice for some time before the introduction of the term.

Northern Blot

The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample.

Introduction

The western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

There are now many reagent companies that specialize in providing antibodies (both monoclonal and polyclonal antibodies) against tens of thousands of different proteins. Commercial antibodies can be expensive, although the unbound antibody can be reused between experiments. This method is used in the fields of molecular biology, biochemistry, immunogenetics and other molecular biology disciplines.

Other related techniques include using antibodies to detect proteins in tissues and cells by immunostaining and enzyme-linked immunosorbent assay (ELISA).

The method originated from the laboratory of George Stark at Stanford. The name western blot was given to the technique by W. Neal Burnette and is a play on the name Southern blot, a technique for DNA detection developed earlier by Edwin Southern. Detection of RNA is termed northern blotting and the detection of post-translational modification of protein is termed Eastern blotting.

Introduction

Southern blotting was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1970s. To oversimplify, DNA molecules are transferred from an agarose gel onto a membrane. Southern blotting is designed to locate a particular sequence of DNA within a complex mixture. For example, Southern Blotting could be used to locate a particular gene within an entire genome. The amount of DNA needed for this technique is dependent on the size and specific activity of the probe. Short probes tend to be more specific. Under optimal conditions, you can expect to detect 0.1 pg of the DNA for which you are probing.

QTL Traits

Polygenic inheritance, also known as quantitative or multifactorial inheritance refers to inheritance of a phenotypic characteristic (trait) that is attributable to two or more genes and their interaction with the environment. Unlike monogenic traits, polygenic traits do not follow patterns of Mendelian inheritance (qualitative traits). Instead, their phenotypes typically vary along a continuous gradient depicted by a bell curve. Though not necessarily genes themselves, quantitative trait loci (QTLs) are stretches of DNA that are closely linked to the genes that underlie the trait in question. QTLs can be molecularly identified (for example, with PCR or AFLP) to help map regions of the genome that contain genes involved in specifying a quantitative trait. This can be an early step in identifying and sequencing these genes.

Genetic Mapping

Perhaps one of the most powerful applications of markers is genetic mapping. This technology allows researchers identify markers located within or very close to genes of interest. With this it is possible to trace these genes through generations of crosses.

If the trait in question is complex and multigenic, mapping will also be of tremendous help to dissect the various genes or loci that contribute to the overall characteristic. Determining the amount of impact each locus delivers is a process called Quantitative Trait Loci or QTL mapping.

Deoxyribonucleic acid (DNA) is a molecule made up of pairs of building blocks called nucleotides. The four kinds of nucleotides that make up DNA are adenine (abbreviated as the single letter A), guanine (G), cytosine (C), and thymine (T). The DNA molecule has the shape of two intertwined spirals, referred to as a double helix. DNA is packaged into chromosomes that are located within the nucleus of all cells. These chromosomes are the same in every cell of an organism and together make up the organism’s genetic information, its genome. Chromosomes contain stretches of DNA called genes that code for amino acids that make proteins. It is the proteins that are the foundation of life for all organisms. The interaction and structure of proteins determine the visible characteristics or phenotype of an organism, while the genetic makeup of an organism is called its genotype.

The sequence of nucleotides that make up a gene can differ among individuals. The different forms of a gene are called alleles. The alleles are the result of nucleotide differences in a gene that affect an amino acid sequence of a protein. This can result in a change, addition, or deletion of a protein that can affect the phenotype.

Silver Staining for Protein

Silver staining is one of the procedures, in addition to Coomassie Blue, R and G types, and fluorescent dyes that are available for detecting proteins separated by gel electrophoresis. Switzer introduced silver staining in 1979, a technique that today provides a very sensitive tool for protein visualization with a detection level down to the 0.3-10 ng level.

The basic mechanisms underlying silver staining of proteins in gels are relatively well understood. Basically, protein detection depends on the binding of silver ions to the amino acid side chains, primary the sulfhydril and carboxiyl groups of proteins, followed by reduction to free metallic silver. The protein bands are visualized as spots where the reduction occurs and, as a result, the image of protein distribution within the gel is based on the difference in oxidation-reduction potential between the gel’s area occupied by proteins and the free adjacent sites. A number of alteration in the silver staining procedure can shift the oxidation-reduction equilibrium in a way that gel- separated proteins will be visualized either as positively or negatively stained bands. Silver staining protocols can be divided into two general categories: 1) silver amine or alkaline methods, and 2) silver nitrate or acidic methods. In general, the detection levels’ using the various procedures is determined by how quickly the protein bands develop in relationship to the background (e.g. signal-to-noise ratio). The silver amine or alkaline methods usually have lower background and as a result are most sensitive but require longer procedures. Acidic protocols, on the other hand, are faster but slightly less sensitive. Clearly, each protocol has different advantages regarding timing, sensitivity, cost, and compatibility with other analytical methods, especially mass-spectrometry (MS) a tool that is being used in combination with gel electrophoresis or chromatographic methods for rapid protein identification. Until recently most of the silver staining protocols used either glutaraldehyde- of formaldehyde-based sensitizes in the fixing and sensitization step, thus, introducing the chemical modifications into proteins. The utilization of those chemicals causes the cross-linking of two lysine residues within protein chain, thus, strongly affecting two most crucial steps for the subsequent MS analysis: hampering the trypsin digestion and highly reduces the protein extraction from the gel .

What are Retrotransposons?

The presence of mobile elements, capable of moving from one region of the genome to another, has been known and thoroughly studied since the 1950’s. These elements are capable of exciding and inserting themselves from one region of the chromatin to another, through their peculiar terminal repeat regions. This process is mediated by enzymes encoded by the transposon itself.

Retrotransposons are a class of mobile elements, widespread in plant genomes, where they account for a significant part of noncoding DNA. Barley, for example, contains thousands of copies of the Bare-1 element, dispersed throughout the euchromatic regions of chromosomes and exhibiting sequence identity higher than 95% to each other.

Unlike other classes of mobile elements, retrotransposons insert into the genome via an RNA intermediate, synthesized by their own reverse transcriptase, without the loss of the parental copy. Unique sequence properties show that retrotransposons of the Ty1-copia group are close to retroviruses, and their mode of integration in the host genome is also very similar.

The number and exact point of insertion of retrotransposons may differ from one individual to another and this can be exploited to characterize individuals, varieties or species, depending on how recent the transpositional event is. Furthermore, insertion of a retrotransposon is an irreversible event, a characteristic that can be helpful when tracing an ancestry.

Single strand conformation polymorphism (SSCP), or single strand chain polymorphism, is defined as conformational difference of single stranded nucleotide sequences of identical length as induced by differences in the sequences under certain experimental conditions. This property allows to distinguish the sequences by means of gel electrophoresis, which separates the different conformations.

Background

A single nucleotide change in a particular sequence, as seen in a double stranded DNA, cannot be distinguished by electrophoresis, because the physical properties of the double strands are almost identical for both alleles. Contrarily, after denaturation, single stranded DNA undergoes a 3- dimensional folding and may assume a unique conformational state based on its DNA sequence. The difference in shape between two single-stranded DNA strands with different sequences can cause them to migrate differently on an electrophoresis gel, even though the number of nucleotides is the same. Predictability, however, is a shortfall of SSCP - since conformational states are subject to many experimental conditions and sequence differences that cause minimal changes in strand conformation may not be detected.

Synonym : PCR-RFLP (PCR- Restriction Fragment Length Polymorphism)

The Cleaved Amplified Polymorphic Sequence or CAPS method is a technique in molecular biology for the analysis of genetic markers. It is an extension to the Restriction Fragment Length Polymorphism (RFLP) method, using polymerase chain reaction (PCR) to more quickly analyse the results.

Like RFLP, CAPS works on the principle that genetic differences between individuals can create or abolish restriction endonuclease restriction sites, and that these differences can be detected in the resulting DNA fragment length after digestion.

In the CAPS method, PCR amplification is directed across the altered restriction site, and the products digested with the restriction enzyme. When fractionated by agarose or acrylamide gel electrophoresis, the digested PCR products will give readily distinguishable patterns of bands. Alternatively, the amplified segment can by analyzed by Allele specific oligonucleotide (ASO) probes, a process that can often be done by a simple Dot blot.

SCARs are DNA fragments amplified by the Polymerase Chain Reaction (PCR) using specific 15-30 bp primers, designed from nucleotide sequences established in cloned RAPD (Random Amplified Polymorphic DNA) fragments linked to a trait of interest. By using longer PCR primers, SCARs do not face the problem of low reproducibility generally encountered with RAPDs. Obtaining a codominant marker may be an additional advantage of converting RAPDs into SCARs. However, SCARs may exhibit dominance when one or both primers partially overlap the site of sequence variation. SCARs are locus specific and have been widely applied in gene mapping studies and marker assisted selection (Paran & Michelmore, 1993).

Since the mid 1980s, genome identification and selection has progressed rapidly with the help of PCR technology. A large number of marker protocols that are rapid and require only small quantities of DNA have been developed. Three widely-used PCR-based markers are RAPDs (Williams et al., 1990), SSRs or microsatellites (Tautz, 1989), and AFLPs (Vos et al., 1995). Each marker technique has its own advantages and disadvantages. RAPD markers are very quick and easy to develop (because of the arbitrary sequence of the primers) but lack reproducibility (Karp et al., 1997; Hansen et al., 1998; Jones et al., 1999; Virk et al., 2000). AFLP has medium reproducibility but is labour intensive and has high operational and development costs (Karp et al., 1997). Microsatellites are specific and highly polymorphous (Karp et al., 1997, Jones et al., 1999), but they require knowledge of the genomic sequence to design specific primers and, thus, are limited primarily to economically important species.

The choice of a molecular marker technique depends on its reproducibility and simplicity. The best markers for genome mapping, marker assisted selection, phylogenic studies, and crop conservation have low cost and labour requirements and high reliability. Since 1994, a new molecular marker technique called inter simple sequence repeat (ISSR) has been available (Zietkiewicz et al., 1994). ISSRs are semiarbitrary markers amplified by PCR in the presence of one primer complementary to a target microsatellite. Amplification in the presence of nonanchored primers also has been called microsatellite-primed PCR, or MP-PCR, (Meyer et al., 1993).