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Minisatellite is a section of DNA that consists of a short series of bases 10– 60bp. These occur at more than 1000 locations in the human genome. Some minisatellites contain a central (or “core”) sequence of letters “GGGCAGGANG” (where N can be any base) or more generally a strand bias with purines (Adenosine (A) and Guanine (G)) on one strand and pyrimidines (Cytosine (C) and Thymine (T)) on the other. It has been proposed that this sequence per se encourages chromosomes to swap DNA. In alternative models, it is the presence of a neighbouring cis-acting meiotic double-strand break hotspot which is the primary cause of minisatellite repeat copy number variations. Somatic changes are suggested to result from replication difficulties (which might include replication slippage, among other phenomena). When such events occur, mistakes are made, this causes minisatellites at over 1000 locations in a persons genome to have slightly different numbers of repeats, thereby making them unique. The most highly mutable minisatellite locus described so far is CEB1 (D2S90) described by Vergnaud.

Also Referred to as Microsatellites

SSR markers are based short sequences of nucleotides (2-6 units in length) that are repeated in tandem:

Di-nucleotide repeat: CACACACA

Tri-nucleotide repeat: ATGATGATGATG

It is believed that when DNA is being replicated, errors occur in the process and extra sets of these repeated sequences are added to the strand. Over time, these repeated sequences vary in length between one cultivar and another.

Introduction

The AFLP amplified fragment polymorphism also known as selective restriction fragment amplification (SRFA) technique is used to visualize hundreds of amplified DNA restriction fragments simultaneously. The AFLP band patterns, or fingerprints, can be used for many purposes, such as monitoring the identity of an isolate or the degree of similarity among isolates. Polymorphisms in band patterns map to specific loci, allowing the individuals to be genotyped or differentiated based on the alleles they carry. produces highly complex DNA profiles by arbitrary amplification of restriction fragments ligated to double-stranded adaptors with hemi-specific primers harboring adaptor-complementary 5' termini.The technique has been widely used in the construction of genetic maps containing high densities of DNA marker loci.

The AFLP protocol amplifies restriction fragments obtained by endonuclease digestion of target DNA using “universal” AFLP primers complementary to the restriction site and adapter sequence. However, not all restriction fragments are amplified because AFLP primers also contain selective nucleotides at the 3' termini that extend into the amplified restriction fragments. These arbitrary terminal sequences result in the amplification of only a small subset of possible restriction fragments. The number of amplified fragments can therefore be “tailored” by extending the number of arbitrary nucleotides added to the primer termini. Alternatively, the use of endonuclease combinations that vary in their restriction frequency can also be used to tune the number of amplicons. Generally, the abundant restriction fragments produced from complex genomes require of AFLP primers with longer selective regions. Conversely, analysis of small genomes require of only few arbitrary nucleotides added at the primer 3' termini. The resulting AFLP fingerprints are usually a rich source of DNA polymorphisms that can be used in mapping and general fingerprinting endeavors.

In 1985, Sir Alec Jeffreys developed restriction fragment length polymorphism (RFLP), which quickly became the standard technique for DNA testing throughout the 1980s. RFLP provided the world with the first form of genetic testing based on DNA, the body’s genetic material.

Definition : RFLP analysis is a difference in homologus DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the samples in question with specific restriction endonucleases (RE).

A restriction fragment length polymorphism , or RFLP, (commonly pronounced “rif lip”), is a variation in the DNA sequence of a genome that can be detected by breaking the DNA into pieces with restriction enzymes and analyzing the size of the resulting fragments by gel electrophoresis. It is the sequence that makes DNA from different sources different, and RFLP analysis is a technique that can identify some differences in sequence (when they occur in a restriction site). Though DNA sequencing techniques can characterize DNA very thoroughly, RFLP analysis was developed first and was cheap enough to see wide application. Analysis of RFLP variation was an important tool in genome mapping, localization of genetic disease genes, determination of risk for a disease, genetic fingerprinting, and paternity testing.

RAPD stands for Random Amplification of Polymorphic DNA. It is a type of PCR reaction, but the segments of DNA that are amplified are random. The scientist performing RAPD creates several arbitrary, short primers (8-12 nucleotides), then proceeds with the PCR using a large template of genomic DNA, hoping that fragments will amplify. By resolving the resulting patterns, a semi-unique profile can be gleaned from a RAPD reaction.

No knowledge of the DNA sequence for the targeted gene is required, as the primers will bind somewhere in the sequence, but it is not certain exactly where. This makes the method popular for comparing the DNA of biological systems that have not had the attention of the scientific community, or in a system in which relatively few DNA sequences are compared (it is not suitable for forming a DNA databank). Because it relies on a large, intact DNA template sequence, it has some limitations in the use of degraded DNA samples. Its resolving power is much lower than targeted, species specific DNA comparison methods, such as short tandem repeats. In recent years, RAPD is used to characterize, and trace, the phylogeny of diverse plant and animal species.

Allele-Specific PCR

This diagnostic or cloning technique is used to identify or utilize single- nucleotide polymorphisms (SNPs) (single base differences in DNA). It requires prior knowledge of a DNA sequence, including differences between 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.

Amplification Refractive Mutation System (ARMS) - PCR

In ARMS, the primer pair is designed so that one of the 3' ends coincides with a variant nucleotide in the target sequence. When the primer mismatches the template the frequency of extension is very low and consequently the effective number of sequence copies available for amplification is greatly reduced. ARMS PCR exploits a thermostable polymerase that lacks 3' exonuclease activity (usually Taq polymerase). Such enzymes extend primers bound to their target sequences very inefficiently when the 3' base is mismatched. Because the 3' exonuclease activity required for mismatch repair is not present, the extension of such primers in PCR is a rare event and amplification is retarded. However, once mismatch extension has occurred amplification proceeds normally from the newly synthesised target strand. Thus an important principle of ARMS is that non-matching (i.e. non-matching at only the 3' end of one primer) template may be amplified. The factors that contribute to this apparent ‘failure’ of ARMS are that when very high numbers of template molecules are added to the reaction, amplification is likely to proceed because the small proportion of mismatched primer extensions reach the sensitivity threshold of the reaction. Second, certain mismatches are extended more efficiently than others so that proportion extended in each PCR cycle varies.

Polymerase Chain Reaction

The Polymerase Chain Reaction (PCR) technique, invented in 1985 by Kary B. Mullis, allowed scientists to make millions of copies of a scarce sample of DNA. The technique has revolutionized many aspects of current research, including the diagnosis of genetic defects and the detection of the AIDS virus in human cells. The technique is also used by criminologists to link specific persons to samples of blood or hair via DNA comparison. PCR also affected evolutionary studies because large quantities of DNA can be manufactured from fossils containing but trace amounts.

Kary Mullis invented the PCR technique in 1985 while working as a chemist at the Cetus Corporation, a biotechnology firm in Emeryville, California. The procedure requires placing a small amount of the DNA containing the desired gene into a test tube. A large batch of loose nucleotides, which link into exact copies of the original gene, is also added to the tube. A pair of synthesized short DNA segments, that match segments on each side of the desired gene, is added. These “primers” find the right portion of the DNA, and serve as starting points for DNA copying. When the enzyme Thermus aquaticus (Taq) is added, the loose nucleotides lock into a DNA sequence dictated by the sequence of that target gene located between the two primers.

The test tube is heated, and the DNA’s double helix separates into two strands. The DNA sequence of each strand of the helix is thus exposed and as the temperature is lowered the primers automatically bind to their complementary portions of the DNA sample. At the same time, the enzyme links the loose nucleotides to the primer and to each of the separated DNA strands in the appropriate sequence. The complete reaction, which takes approximately five minutes, results in two double helices containing the desired portion of the original. The heating and cooling is repeated, doubling the number of DNA copies. After thirty to forty cycles are completed a single copy of a piece of DNA can be multiplied to hundreds of millions.

Restriction Enzymes

Restriction enzymes are enzymes isolated from bacteria that recognize specific sequences in DNA and then cut the DNA to produce fragments, called restriction fragments. Restriction enzymes play a very important role in the construction of recombinant DNA molecules, as is done in gene cloning experiments. Another application of restriction enzymes is to map the locations of restriction sites in DNA.

Restriction endonucleases, often called restriction enzymes, cut DNA within the molecule by hydrolyzing the phosphodiester bonds between the nucleotides. Restriction enzymes were discovered in bacteria and there are now more than 1200 known restriction enzyme types. These enzymes are named using a simple system. EcoRI, for example, was isolated from E. coli and was the first enzyme isolated from a particular strain, hence the designation of I. HaeIII was isolated from Haemophilus aegyptius. HindIII was isolated from Haemophilus influenzae, and was the third enzyme discovered in a particular strain.

Usually, restriction enzymes only cut the DNA at or near a very specific nucleotide sequence known as a recognition site. This “restrictive” nature of these enzymes allows molecular biologists to pin-point exactly where the DNA is to be cut. In many cases, the recognition site of a restriction enzyme consists of a palindromic sequence where the order of the bases is read the same on each side of the helix. The two sides of the helix are antiparallel and the recognition sequence reads from 5’ to 3’ on either side of the helix. For example the recognition site for EcoRI is as follows;

Agriculture Scenario

It has been seen since last few decades there is huge decrease in the production of the agricultural production. Three factors which can be held responsible for the increase of agricultural production per hectare:

1. Overall increase in efficiency due to the merger of farms and a more efficient redistribution of farm land.
2. More efficient cultural practises and better quality and availability of inputs (fertilizer, pesticides).
3. Genetic improvement of crops.

At this moment over 6 billion people need to be fed and it is expected that in the year 2050 more than 10 billion people will inhabit the earth (FAO, 1996). Moreover, demands per capita will rise when standards of living in developing counties improve. As a consequence there will be a huge increase in the demand for food, and production will need to triple in the coming 40 years. A continued effort to secure agricultural production in the future will therefore be vital. Reduction of losses caused by a-biotic and biotic stress will be a key issue. Scenario studies on world food security generally assume that a large increase in production will be achieved by genetic improvement of crop species and biotechnology. History has indeed shown a continuous increase in crop yields, resulting from plant breeding efforts. However, in the light of the speed at which the human population develops, and taking into account the expected reduction of available arable land due to climate change and human intervention, it may be necessary to accelerate the rate at which genetic improvement is achieved. Modern biotechnology provides new tools that can facilitate development of improved plant breeding methods and augment our knowledge of plant genetics. The knowledge that is obtained with these new tools can be used to contribute to an enhanced food security throughout the world.

There are many different methods of extracting DNA bands from an agarose gel. Which one you choose will probably depend on the consumables you have available in your lab. Another important consideration is the yield/ purity of the DNA after extraction. Agarose contains various impurities which may inhibit downstream reactions if not efficiently removed from the DNA. We used to give the generic term ‘hoonose’ to the unspecified carbohydrates that co-purified along with the DNA and inhibited enzyme reactions. Modern spin-column kits are very good at removing impurities, old-fashioned ‘kitchen sink’ methods are less good.

Cutting Out the DNA Band

Most of the methods require you to cut out the band. You must visualise the band, in an ethidium bromide stained gel, in a dark-room on a UV light- box (a trans-illuminator). UV is dangerous, wear gloves, long-sleeves and face protection. Never look at UV with unprotected eyes. If possible, set the trans-illuminator to long-wavelegth UV (or low-power) and minimise the amount of time the DNA is exposed. This is because the UV mutagenises the DNA at a measurable rate. Use a scalpel blade to cut around the band of interest. Switch off the transilluminator, switch on the white light and carefully remove the band from the gel and place it on the glass. It is good to trim off as much empty agarose as possible so go back to UV illumination briefly to do this. Place the excised band in a 1.5mL microfuge tube.

Hunter and Mohler discovered the isozymes. In 1959 Markert and Moler introduced the concept of isozymes, which they defined as the different molecular forms in which proteins may exist with the same enzymatic specificity (Buth 1984). This means that different variants on the same enzymes have identical or similar functions and are present in the same individual. As such, their importance for understanding gene action in development and differentiation was exploited during the1960s in both animals and plants. Nevertheless, isozymes played a minor role in research on plant biochemistry until 1966 when genetic polymorphism for isozymes within the same population was discovered (Stebbins 1989, Wendel 1989). That revealed the possibility for population genetics to make precise quantitative estimates of genetic variability based upon one parameter of the molecular structure of the primary products of the genes themselves.

Plant population genetics were not long in following their zoological colleagues and the investigation of both animals and plants increased explosively. This application, however, necessitated a partitioning of the isozyme concept, since the relevant isozyme subset, defined by Prakash (Buth 1984) as the variant proteins produced by allelic forms of the same locus, to avoid the now common confusion with isozymes which are the various polymer produced from monomers specified by different loci. The development of genetic distance or similarity coefficients (Lukasová 1985) allowed the summarization of isozyme data for intersample comparisons and so quantified isozyme data were soon applied to comparative studies of taxa.

Proteins

Proteins are of central importance in the structure and function of all living organisms. Some proteins serve as structural components while others function in communication, defense, and cell regulation. Some proteins serve as enzymes that act as biological catalysts which control the pace and nature of essentially all biochemical events. Although DNA serves as the genetic blueprint of a cell, none of the life processes would be possible without the proteins.

Electrophoresis of Proteins

Proteins can be separated and purified. Methods for separating proteins take advantage of properties such as charge, size, and solubility, which vary from one protein to the next. Because many proteins bind to other biomolecules, proteins can also be separated on the basis of their binding properties. The source of a protein is generally tissue or microbial cells. The cell must be broken open and the protein must be released into a solution called a crude extract. If necessary, differential centrifugation can be used to prepare subcellular fractions or to isolate organelles. Once the extract or organelle preparation is ready, a variety of methods are available for separation of proteins. Ion-exchange chromatography can be used to separate proteins with different charges (similar to the way amino acids are separated). Other chromatographic methods take advantage of differences in size, binding affinity, and solubility. Nonchromatographic methods include the selective precipitation of proteins with salt, acid, or high temperatures. In addition to chromatography, another important set of methods is available for the separation of proteins, based on the migration of charged proteins in an electric field, a process called (gel) electrophoresis. Gel electrophoresis is especially useful as an analytical method. Its advantage is that proteins can be visualized as well as separated, permitting a researcher to estimate quickly the number of proteins in a mixture or the degree of purity of a particular protein preparation. Also, gel electrophoresis allows determination of crucial properties of a protein such as its isoelectric point and approximate molecular weight.

Polyacrylamide

Polyacrylamide is a cross-linked polymer of acrylamide. The length of the polymer chains is dictated by the concentration of acrylamide used, which is typically between 3.5 and 20%. Polyacrylamide gels are significantly more annoying to prepare than agarose gels. Because oxygen inhibits the polymer- ization process, they must be poured between glass plates (or cylinders).

Two types of molecular weight (MW) standards are routinely used. The Lambda/HindIII and PhiX174/HaeIII MW standards provide a useful reference for calculating molecular weights of large and small DNA fragments, respectively, after electrophoretic separation; the “internal MW standards” provide a means for normalizing fragment migration distances within each lane to facilitate comparisons between lanes on the same or different luminographs in fingerprinting studies.

Electrophoresis is an analytical method frequently used in molecular biology and medicine. It is applied for the separation and characterization of proteins, nucleic acids and subcellular-sized particles like viruses and small organelles. Its principle is that the charged particles of a sample migrate in an applied electrical field. An electric current is passed through a medium containing the molecules, and each molecule travels at a different rate depending on its electrical charge, size and shape. Separation by electrophoresis is based on these differences. In electrophoresis, agarose and acrylamide gels are used for electrophoresis of nucleic acids and proteins.

What is Gel Electrophoresis?

A gel is a solid form. The term electrophoresis describes the migration of charged particle under the influence of an electric field. Electro refers to the energy of electricity. Phoresis, from the Greek verb phoros, means “to carry across.” Thus, gel electrophoresis refers to the technique in which molecules are forced across a span of gel, motivated by an electrical current. Activated electrodes at either end of the gel provide the driving force. A molecule’s properties determine how rapidly an electric field can move the molecule through a gelatinous medium.

Although it can be argued that due to its overall trendiness, they have been a lot of recent developments in making it more fun to work with. As a molecule, it is very similar to DNA (the most notable chemical difference is its flexibility), but suffers because the nucleases (RNases) that can destroy it are notoriously very stable and very difficult to get rid of.

What is DNA

DNA is a simple molecule

DNA is negatively charged

DNA has lots of ring structures; ring structures are generally quite hydrophobic.

DNA is a very robust molecule.

DNA work has lots of toys.

1. pH meter

pH meter/electrode systems are the most common method of measuring pH in the biology laboratory. Although more expensive and more complicated to use than chemical indicators, pH meters provide greater accuracy, sensitivity, and flexibility. When used properly, pH meters can measure the pH of a solution to the nearest 0.1 pH unit or better, and they can be used with a variety of samples.

The concept of gene has undergone several refinements since its discovery in 1866 by Mendel. Most genes encode one polypeptide and can be operationally defined by the complementation test. The complementation or cis-trans test provides an operational definition of the gene. It is used to determine whether mutations are in the same gene or in different genes. Intragenic complementation may occur when a protein is a multimer containing at least two copies of one gene product. The concept of gene has evolved from a bead on a string, not divisible by recombination or mutation to a sequence of nucleotide pairs in DNA encoding one polypeptide chain. The unit of genetic material not divisible by recombination or mutation is the single nucleotide pair. Historically several experimental developments have occurred to facilitate understanding of the structure and function of gene and these are briefly described in a chronological order as follows to have better understanding of the gene concept as it evolved. However, before discussing evidences supporting various gene concepts, it is better to summarize important stages in the evolution of the gene concept.