Nanotechnology

"Next big thing is small"
The word ‘nano’ meaning ‘dwarf’ in Greek language refers to dimensions on the order of magnitude of 10-9. Nanotechnology, focusing on special peoperties of materials emerging from nanometer size, for e.g. In biological systems, occurs at the nanoscale structures where all the fundamental properties and functions are systematically defined “Purposeful engineering materials at nano scale- Nanotechnology”
Nanotechnology as it impacts fields of electronics and information, biology, chemistry and medicine, energy, the environment, transportation, and especially that of materials, is briefly considered.
Nanoparticles:
Three dimensions in the nm range – the three dimensions are of similar size i.e. particles approximate to being spherical. Examples include polymer particles generated by emulsion polymerisation, by polymerisation of micelles, vesicles etc.
Two dimensions are of similar size and are in the nm range whereas the third is longer.
Examples include oblate/acicular particles, nanotubes (carbon, protein) or whiskers (boron nitride, cellulose). One dimension in the nm range with the other two dimensions being longer. Examples are sheets of material which are a few nm thick but may be 100 to 1000nm long. Materials having such structures are layered silicates (phyllosilicates) Particles having three dimensions in the nm range.
Routes and Means:

Micelles:

• Micelles have been used as micro reactors.
• Generation of small metallic particles e.g. gold, silver, platinum. Generation of nm particles of semiconducting sulfides etc. These particles possess very useful luminescent properties–Quantum dots. These dots should find application in security devices and as biological markers.
• Polymersiation of micelles to generate nanospheres (useful for controlled release of drugs or “marker” materials).

Particles generated by sol-gel chemistry:

• Titanium dioxide particles stabilised by means of a protective colloid have been prepared and shown to possess special luminescent and catalytic properties.
• Silica particles have been produced. These have been generated possessing reactive groups on the surface which allows them to be polymerised into a coating (e.g. (meth)acrylate groups – Hanse Chemie GmbH). The particles have very little effect upon the clarity or the viscosity of formulations. The cured coatings possess improved scratch and abrasion resistance, toughness and hardness. Polymerisable monomers such as acrylates, containing derivatised silica particles are available from Clariant France. Others functionalised silica particles have been generated which allow controlled polymer growth via a free radical mediated process.
• Silica and titanium dioxide particles for near infra-red reflective coatings. Coatings having refractive indices from 1.47 to 1.93 have been generated.
  Gold citrate sols for generation of nm dots of gold (via electron beam radiation) for nm circuitry.

Hyperbranched Polymers:

Hyperbranched polymers may be considered as imperfect dendrimers since they do not possess a unique molecular weight but nevertheless have a polydispersity close to unity. Some of these materials are now commercially available. Perstorp Speciality Chemicals have an aliphatic polyester which can be rendered polymerisable.
Uses:
Generation of surface coatings
Production of nanoporous organosilicates (low dielectric constant materials – nano and microcircuitry).

Emulsion polymerization:

• Controlled chemical reaction .
• Generation of dyes and pigments by means of a microreactor (Clariant GmbH). Such particles are suitable for use as ink jet colourants. Hybrid polymers based on polyhedral oligomeric silsesquioxanes POSS. The silsesquioxane unit, which has an outer diameter of 1.5 nm has been incorporated into polymers to impart greater thermal and mechanical stability to the polymer.

Titanium dioxide:
Titanium dioxide for sun screens (ICI) - ~50nm.
Titanium dioxide for fibre treatment (BASF) - ~ 500nm
Three scenarios
A nano sized matrix containing smaller crystallites
True nanoparticles of titanium dioxide can be generated by sol-gel chemistry. These particles show photocatalytic properties and analysis shows that the oxide is of the anatase modification. A micron sized matrix containing nano sized crystallites.
Precipitation of titanium dioxide from its alkoxides appears to give an amorphous powder which in fact contains nano and sub-nano sized crystallites of anatase. When this material is heated crystallization occurs and the crystallites grow in size. The particles produced may have dimensions of several microns but the anatase crystallites may still have nanometer dimensions.
True nanosized particles as used in sunscreens (ICI)
Some current uses and developments:
Oxonica – nanosized phosphors for electronic displays, catalysts, cosmetics and quantum dots.
QinetiQ – use of plasma processing technology for production of nanoparticles by the tonne.
Enact Pharma – nano sized structures for tissue growth
Nanoco – manufacture of quantum dots (samples available on request!). Materials could be of great value for security devices including printing of currency.
Carbon nanotubes – electronics (transistors, field emitters), electrically conducting paints.
Nanotubes:
These structures are found in nature e.g. cellulose and self-assembling proteins. Designer protein nanotubes have the potential to kill bacteria which are resistant to antibiotics e.g. Staphylococcus aureus. Undoubtedly, as far as composites are concerned, carbon nanotubes offer the greatest potential. They are formed from sheets of graphite which are rolled up into tubes having a diameter of a few nanometers. Depending on the way the sheets are rolled up, the nanotubes may either be semiconducting or conducting. The semiconducting material has been used to make nm sized Field-effect transistors. Conducting carbon nanotubes can render paints conducting when incorporated at the 2% level. This is of great potential benefit for electropolymer design. Nanotubes being commercially produced have ~10nm diameter and are composed of layers of graphite. Carbon nanotubes are 200 times stronger than steel and now that they can be produced in lengths up to 20cm they should be of great value as reinforcing materials for automobile components where their lightness will benefit fuel economy. Another advatage is that the nanotubes can be used at such a low loading that they do not interfere with the mechanical properties of the composite.

One dimension in the nm range –layered silicates

The layered silicates are being used as nano composites. They offer the following:

• Efficient reinforcement with minimal loss of ductility and impact strength.
• Thermal endurance.
• Flame resistance.
• Improved barrier properties.
• Improved abrasion resistance.
• Improved chemical resistance.
• Reduced shrinkage and residual stress.
• Altered electrical, electronic and optical properties.
• Their use to improve scratch, mar and abrasion resistance.
• Use of their barrier properties to make more ecofriendly packaging with the possibility of reducing our dependence upon laminates.
• To utilise their effectiveness in reducing the flammability of coatings thereby aiding compliance with new EC regulations.
• Improving the strength and durability of coatings.

Structure :
The layered silicates commonly used in nanocomposites are the 2:1 phyllosilicates. Their crystal lattice consists of two-dimensional layers where a central octahedral sheet of alumina or magnesia is fused to two external silicate layers. The layers organise themselves to form stacks with a regular van der Walls gap between them called an interlayer or gallery. Isomorphic substitution within the layers e.g. Al3+ by Mg2+ generates negative charges that are counterbalanced by alkali or alkaline earth cations. The forces holding the stacks together are relatively weak and hence intercalation of small moleucules between the layers is easy. The phyllosilicates can be rendered organophilic by exchanging the sodium ions for alkylammonium ions. Layered silicates may be incorporated into polymers in three different ways: Intercalation of polymers Intercalation leads to a single extended polymer chain being threaded through the gallery.
Direct intercalation by mixing the silicate and the polymer e.g. poly (ethylene oxide). Most polymers are incompatible with the silicate making this method unsuitable.
Exchange of the sodium ions in the gallery by means of alkylammonium salts followed by mixing with the polymer e.g. poly(styrene), polyamides, polyesters, polycarbonates, polyphosphazenes and polysiloxanes. Intercalation has been achieved by mixing the modified silicate with polymers in the melt e.g. poly(styrene), poly(propylene) modified with maleic anhydride, ethylene-vinyl acetate copolymers, poly(styrene-b-butadiene) copolymer.
Intercalation of polymer precursors e.g. 12- aminolauric acid - caprolactam, e-caprolactone, or a reactive component e.g. diglycidyl ether of bisphenol A followed by reaction with multifunctional amines, (vinlybenzyl)trimethylammonium chloride.
Template synthesis: Intercalation of polymerization initiators such as Ziegler-Natta catalyst for polyolefin production, photoinitiators and initiators for controlled free radical polymerization e.g.
The Impact of Nanotechnology in Drug Discovery:
Pharmaceutical and biotechnology companies are under immense pressures to produce a steadystream of innovative, well-differentiated drugs at reduced costs. Currently it takes an estimated 7-10 years to develop and market a drug at a cost that exceeds $800 million. In simple terms, drugdiscovery requires the identification of a disease, knowledge of the disease mechanism and identification of a target (point of intervention).The human genome project is expected to identify approximately 100,000 targets that will require evaluation against many compound libraries to compare gene sequences and structure. This represents a very time-consuming process and a major bottleneck in the drug discovery process as millions of compounds can be screened for each target. Novel discovery and validation technologies can expand the hit rate for promising compounds in the pipeline, and expedite their progress through to market. The introduction of microarrays and lab-on-a-chip technologies has already revolutionized the drug discovery process. Where once it took the effort of a single chemist to view anything from one to 12 gene variations at a time, microarrays can view thousands in the same time frame. Now, nanotechnology promises to exponentially increase even the volume of microarrays by working at a level far smaller than conventional microarrays and according to one source the pharmaceutical market will represent
approximately $180 billion of the forecasted $1 trillion nanotech industry. Nanotechnology can enhance the drug discovery process, through miniaturization, automation, speed and reliability of assays. Although at an embryonic stage of development, nano-enabled drugs are already bringing clinical benefits to thousands of patients. This report provides an overview and a detailed analysis of the major drivers, restraints and challenges for current and emerging applications of nanotechnology in drug discovery. Areas covered include nano-enabled drug discovery technologies/tools, nano-enabled drugs and an assessment of key market engineering parameters. The technologies/tools covered include, nanoarrays, nanomasspectrometry, nanolithography arrays, biological chips, congruent force
intermolecular test and solubility and reformulation methods. This report provides global market coverage and discusses funding, regulatory and ethical issues in the major industrial countries, with worldwide market forecasts over a 5-year forecast period. This report will be of vital interest to strategic planners, marketing managers and product development managers at all pharmaceutical, diagnostic, life science and biotechnology companies that may be interested in nanotechnology. Currently the estimated market for nanoparticles is valued at ~£27,000m a year of which ~23% is concerned with nanocomposites.
Improvement in mechanical properties through the use of nanocomposites:
Examples include:
Nylon nanocomposites where addition of 4 wt. % alumino-silicate creates a substantial increase in strength and modulus and this is not accompanied by a decrease in impact resistance.
Silicate-epoxy nanocomposites. 4 wt. % incorporation led to a 60% increase in storage modulus in the glassy region and in the rubbery region a 450% increase was observed. Other epoxy nanocomposites having a sub-ambient Tg exhibited a 10 fold improvement in modulus.
Nylon-6 nanocomposites show a drastic increase in Young’s modulus at low filler content. Improvements in mechanical properties are dependent upon the nature of the interactions between the matrix and the filler (intercalative versus exfoliated structures, ionic interactions as determined by the structure of the alkylammonium salt, the polarity of the polymer). Thermal stability and flame retardant properties. Thermal stability.

Examples include:

• PMMA at 10 wt.% loading resisted degradation under conditions where PMMA itself is completely degraded.
• Polydimethylsiloxane (a 1400 C higher decomposition temperature achieved.
• Polyimide (magnitude depend upon whether clay is intercalated or exfoliated).
• Poly (styrene) (effectiveness dependent upon type of alkylammonium compound used).

Flame retardent properties:

• Two main classes of “flame-safe” coatings.
• Flame retardent coatings (delay ignition and flame spread).
• Intumescent coatings (delay ignition and flame spread and also protect the substrate by forming an insulation layer during combustion. The multilayered silicate appears to act as an excellent insulator and mass transport barrier thereby slowing down the escape of volatile products generated on pyrolysis. The heat release ratio is much lower for nanocomposites compared with the unmodified polymers.

Examples of flame retardency:

• Delaminated nylon-6 and nylon-12 nanocomposites.
  Poly(styrene).
• Ethylene-vinyl acetate copolymer
  Poly(caprolactone) .
• An intumescent nanocomposite based on Nylon-6 and ammonium polyphosphate has been produced.

Nano nutrition for plants :
Nano nutrients are highly efficient interms of plant uptake mobility and utilisation. Nano particles of ZnO, FeO, CuO, MnO, MgO and CaO with different sizes were synthesised and their formulations were designed, for enhanced plant growth and yields.

Tissue Culture Methods

The MS (Murashige and Skoog) medium is prepared by making stock solutions of various chemicals (both macro nutrients and micro nutrients) and later mixing them in required proportions in the distilled water to get desired concentrations.
Preparation of stock solutions of macro and micro nutrients of MS medium:
i) Stock solution A: 16.5 g of ammonium nitrate (NH4N03), 19 g of Potassium nitrate (KN03), 3.7 g of Magnesium sulphate (MgS04 7H20) and 1.7 g of Potassium dihydrogen phosphate (KH2P04) are weighed and dissolved in about 300 ml of distilled water. Then the final volume was made up to 500 ml with distilled water.
ii) Stock solution B: 41.5 mg of Potassium iodide (KI), 310 mg of Boric acid (H3B03), 111.5 mg of Manganese sulphate (MnS04 4H20), 430 mg of Zinc sulphate (Zn S04 7H20) and 12.5 g of Sodium molybdate (Na2 Mo04 2H20) were weighed and dissolved in 50 ml distilled water. 1.25 ml of stock solution of Copper sulphate (CuS04 5H20) and Cobalt chloride (CoCl2 6H20) is added. The final volume is made to 100 ml by adding distilled water.
Stock solutions of copper sulphate and cobalt chloride: 100 mg of Copper sulphate (Cu S04 5H20) and 100 mg of Cobalt chloride (CoCl2 6H20) is accurately weighed and dissolved in 50 ml distilled water. Then the final volume is made to 100 ml by adding distilled water.
iii) Stock solution C: 373 mg of Ethylene diamine tetra acetic acid disodium salt (Na2 EDTA) is weighed and dissolved in about 50 ml distilled water by constant heating and stirring. Then 278 mg of Ferrous sulphate (FeS04 7H20) is weighed and added slowly to the same solution with constant heating and stirring until completely dissolved. The final volume is then made to 100 ml with distilled water.

iv) Stock solution D: 200 mg Glycine, 50 mg of Nicotinic acid, 50 mg of Pyridoxine HCI and 10 mg of Thiamine- HCI are weighed and dissolved in 50 ml distilled water and the final volume is made to 100 ml by adding distilled water.
Preparation of stock solutions of hormones:
The Stock solutions (l mg/ml) of various hormones are prepared as follows-
i) Stock solutions of NAA and 2,4D : 100 mg powder of NAA or 2,4D is weighed into a 100 ml volumetric flask and dissolved in 2 to 5 ml of 1.0 N Sodium hydroxide (NaOH). After complete dissolution, the final volume is made to 100 ml by adding distilled water.
ii) Stock solutions of kinetin and benzyl adenine: 100 mg powder of BA or kinetin is weighed into a 100 ml volumetric flask and dissolved in 2 to 3 ml of 1.0 N Hydrochloric final volume is made to 100 ml by adding distilled water.
iii) Stock solutions of IAA and IBA: 100 mg powder of lAA or IBA weighed into a 100ml volumetric flask and dissolved in few drops of 90 per cent Ethyl alcohol. After complete dissolution, the final volume is made to 100 ml by adding distilled water.
All the stock solutions are stored in amber colored bottles and placed in refrigerator. The required quantities are pipetted out at the time of media preparation. The volume of stock
Steps followed during media preparation:
The medium can be prepared in glass beaker of 1000 ml and 2000 ml capacity depending on the requirement. The following steps are followed during the preparation of (one litre) medium:
1.30 g of Sucrose is weighed and dissolved in approximately 300 ml distilled water in a glass beaker.
2. 50 ml of stock solution 'A', 2 ml of stock solution 'B', 10 ml of stock solution 'C' and 1 ml of stock solution 'D' are added in sequence.
3. 440 mg of CaCl2 2H20 and 100 mg of Myo - inositol are added directly.
4. Required concentrations of hormone (s) are added to the medium and the volume is made approximately 650 ml.
5. 8.0 g of agar-agar is boiled in approximately 300 ml distilled water and added to the prepared solution.
6. The pH of the medium is adjusted to 5.8 with 0.1 N Sodium hydroxide (NaOH) or O.1N Hydrochloric acid (HCI) as needed and the volume is made up to 1.0 litre.
7. The medium is dispensed in culture vessels at the rate of 15-20 ml per tube or 25 to 30 ml per petri plate and the tubes are closed tightly with cotton plugs.
8. The culture vessels along with medium are autoclaved for 20 minutes after the pressure reached to 1.06 kg/cm2 at 121°C for sterilization.
9. The pressure is brought to normal slowly and the culture vessels are taken out of the autoclave and the tubes are kept for cooling in slanting position to get more surface area for explant inoculation. The culture vessels with medium are stored in sterile control room until use.
Surface sterilization of explants:
Mature seeds of the genotypes under study are surface sterilized with 70% ethanol for one minute followed by 0.1 % mercuric chloride for 10-15 minutes. Later the excess mercuric chloride is removed by rinsing the treated explants with sterile distilled water for 4-5 times. After rinsing, the seeds are blotted on sterile paper towels.
Germination of aseptic seedlings:

Filter paper boats are prepared and inserted into culture tubes and tightly closed with cotton plugs. These tubes were sterilized by autoclaving at 121°C and 15 lbs pressure for 20 minutes. In each culture tube, 15 ml of sterile water was poured under the laminar air flow and sterilized seeds were inoculated in culture tubes at the rate of 2-4 seeds/tube depending on the size of the seed and kept for germination under aseptic conditions.
Inoculation:
From the aseptically raised seedlings, explants Viz., root, hypocotyls, cotyledonary leaf, shoot tip etc., are excised with sterile scalpel blade under laminar air flow and used for inoculating in the corresponding callus induction medium.Cultures were scored for callus induction frequency at end of 4th week.
The morphological appearances of the callus is described in terms of its colourd and structure at 40 days after inoculation.

Types of Molecular Markers

INTRODUCTION

Mapping a marker or a mutation to a well defined chromosomal region is an essential step in the genetic analysis of a plant and is also (unless the mutant is tagged) a prerequisite for molecular cloning of the corresponding gene. Determining the map position of a marker or a gene (as identified by its mutant phenotype) consists in testing linkage with a number of previously mapped markers. Once linkage with a specific marker is detected, a refined mapping can be achieved by analysing linkage relations to more markers in that region.
Historically, mapping in plants primarily utilized morphological markers such as mutants with an easily scorable phenotype and a defined map position. Typically, the mutant of interest is crossed to another mutant used as phenotypic marker, the resulting F1 double heterozygote is allowed to self, and segregation of the two phenotypes is analysed in the F2 population.

The mutation used as marker should of course not interfere with the phenotype of the mutant to be mapped. The genetic distance is the number of meiotic recombination events that occur between the two loci in 100 chromosomes. The genetic distance is expressed in centiMorgans (cM), and can range from 0 cM (absolute linkage) to 50 cM (non-linked loci). It remains however difficult to score many different phenotypes in a single population. Hence, detailed mapping using morphological markers is tedious because it requires numerous crosses.
In contrast, a single cross can be used to analyse linkage with an essentially unlimited number of molecular markers. DNA markers were incorporated into mapping strategies once it was recognized that distantly related individuals differ in DNA sequence throughout their genome [Botstein, 1980]. Molecular markers exploit the natural differences between distinct lines. For instance, in Arabidopsis, it has been estimated that the widely used Landsberg erecta and Columbia ecotypes differ by approximately 0.5 to 1 % at the DNA sequence level [Chang, 1988; Hauser, 1998]. These local differences or polymorphisms of the DNA sequence are due to point mutations, insertion or deletions that randomly occurred in one ecotype and not in the other. These DNA polymorphism can be conveniently visualized by several methods.

To map a novel mutation that was generated in line A, this mutant is crossed with a wild-type plant of a polymorphic line B, and the Fl progeny is allowed to self. The resulting F2 population can then be used to Self. The resulting between the mutation of interest and any DNA marker that distinguished line A and line B. As compared to Morphological markers, an additional advantage of molecular markers is that in most cases homozygous and heterozygous individuals can be readily distinguished.
In addition, data will be generated using M. truncatula Recombinant Imbred Lines (RKLs) in combination with the SSR ( Microsatellites) method. Data will be analyzed using the MAPMAKER program or the colormapping procedure.
DIFFERENT TYPE OF DNA MARKERS
· RFLP
· CAPS
· RAPD
· AFLP
· SSR
· SNPs
The types of markers that will used during the practical course are indicated with. However, other types of DNA markers that can be used for genetic mapping are also described. By definition we call "DNA marker" a DNA fragment which exhibit polymorphism between two lines.

Restriction Fragment Length Polymorphism (RFLP)
The first type of DNA markers that were used for genetic mapping were RFLPs. The DNA sequence differences between polymorphic lines may create differences in the length of restriction fragments derived from genomic DNA. For instance a given restriction site may be present in one line and not in the other. This polymorphism can be revealed by genomic DNA blot hybridization (Southern) using as probe a DNA fragment corresponding to that region. The polymorphic bands can then be used as genetic markers to distinguish the two lines. Multiple RFLP markers can be identified and assembled into genetic maps.
An advantage of the RFLP mapping approach is that RFLP markers are co-dominant. Distinct patterns are indeed obtained for plants that are homozygous or heterozygous for the parental alleles. Hence, an the chromosomes of a given F2 population can be scored. In contrast, a main disadvantage is that RFLP mapping necessitates relatively large amounts of DNA because distinct RFLP markers may require digestion of genomic DNA samples with different diagnostic restriction enzymes.

Cleaved Amplified Polymorphism Sequences (CAPS)
The principle of CAPS markers is very similar to that of RFLP markers. The main difference is that PCR is used instead of DNA blot hybridisation to detect a restriction site polymorphism. A genomic DNA region is amplified by PCR using specific primers and those amplified fragments are then digested with diagnostic restriction enzymes to reveal the polymorphism. Hence, whereas RFLP probes can be anonymous colnes, CAPS markers require sequence information to design the specific PCR primers.
Like RFLPs, CAPS markers are co-dominant. CAPS markers are based on PCR for detection, and thus require only small quantities of genomic DNA. Typically a single leaf will provide enough DNA for analiysis with multiple CAPS markers.
Finally, CAPS markers can be easily assayed using standard agarose gel electrophoresis.

Random amplified polymorphic DNA (RAPD)
RAPD markers are another type of PCR-based markers that have been used for genetic mapping [Wiliams, 1993]. This approach is based on the amplification of random DNA segments with single primers of arbitrary nucleotide sequence. The oligonucleotide (around 10­bp long) is used for PCR at low annealing temperatures. When the oligonucleotide hybridises to both DNA strands at sites within an appropriate distance from each other, the DNA region delimited by these two sites will be amplified. Small nucleotide changes (polymorphism) at one of the two sites may prevent hybridization of the oligonucleotide and hence also prevent DNA amplification [Williams, 1990). Typically a RAPD primer will amplify a given fragment from Line A and not from line B. It will impossible to distinguish an homozygous individual AA from an heterozygous individuals AB. In other words, RAPDs are dominant markers and are thus less efficient than co-dominant markers in extracting information from a given F2 population. Another limitation of RAPD marker is that because of the low annealing temperatures used, the amplification of a given polymorphic band seems to be highly sensitive to PCR conditions and hence less consistently reproducible in different laboratories.
Amplified restriction Fragment Length Polymorphism (AFLP)
AFLP TM is a patented technology developed by Key Gene, Wageningen, the Netherlands [Vos et al.,1995]. In this procedure, the genomic DNA is digested by two different restriction enzymes, a rare cutter and frequent cutter. Double-stranded adapters are then ligated to the ends of the restriction fragments. The fragments are then amplified by PCR using primers that correspond to the adapter and restriction site sequences. These primers have additional nucleotides at the 31 ends extending into the restriction fragments, in order to limit the number of fragments that will be amplified. The AFLP products are detected by labeling one of two primers, and lablled DNA fragments are separated by electrophoresis in denaturing polyacrylamide gels (similar to sequencing gels). Typically, 50 to 100 amplification products are detected in a single lane. polymorphic bands can be identified by comparing the amplification products derived from two lines. Like RAPDs, AFLPs are typically dominant markers.
Simple sequence repeats (SSR)
Like other eukaryotic genomes, the plant genome contains tandem repeats of one- two or three-nucleotide motifs. These microsatellite repeat sequences are usually polymorphic in different lines because of variations in the number of repeat units. These polymorphisms are called SSR, and can be conveniently used as co-dominant genetic markers. Specific primers are used to PCR amplify a small genomic region (150 to 250 bp) that contains a polymorphic microsatellite sequence. The size of the amplified fragment will vary depending on the number of repeats present in a given line. These polymorphic fragments can be separated and visualized by electrophoresis in agarose or polyacrylamide gels.
As compared to CAPS markers, SSR offer the additional advantage that they do not involve the use of restriction endonucleases and thus avoid the problems associated with partial digestions.
Single Nucleotide Polymorphisms(SNPs)
The most common class of DNA polymorphisms present both in natural lines and after induced mutagenesis is single nucleotide polymorphisms (SNPs). The RFLP and CAPS methods can detect only the SNPs which alter a recognition site for a restriction enzyme. The RAPD and AFLP methodologies can in principle detect any type of SNPs, however these two techniques are not very convenient to target a selected genomic region. In contrast, plant genome sequencing is generating a wealth of sequence information which provides a starting point for the development of PCR-based markers. In other words, once the sequence of a region is known, primers can be synthesized to amplify alleles of interest which can then be analyzed to find allele-specific polymorphisms in that region.
Although they have not been extensively used in plants thus far, a large number of techniques have been developed to scan a defined region of DNA for SNPs reviewed by Cotton, 1997. In particular, single-strand conformation polymorphism (SSCP) is based on the fact that a strand of single-stranded DNA folds differently from another if it differs by a single base, which leads to different motilities of these two strands in non-denaturing gel electrophoresis. Heteroduplex analysis is based on the different motilities of homo- and heteroduplexes in non-denaturing gel electrophoresis, or in slightly denaturing high-performance liquid chromatography. It should be noted that methods to detect SNPs can also be extremely useful in the step of positional cloning of a mutant locus, namely to locate the mutation within the DNA region has been delimited by mapping.
Finally, an efficient method has been described that allows to create a PCR-base marker for any known point mutation. This technique is called derived cleaved amplified polymorphic sequence (dCAPS), and has been recently applied to Arabidopsis (Michaels 1998; Neff, 1998). The dCAPS method is primarily used when the point mutation of interest does not alter an existing restriction site. In this case, the dCAPS technique consists in designing a primer with one or two mismatches which, together with the mutation, will create a unique restriction site in one only of the two alleles. A second primer (usually without mismatch) is used to PCR amplify the region, and the amplification products are digested with the appropriate restriction enzyme, exactly as for CAPS markers. dCAPS are useful for genetic mapping, and to follow known mutations in segregating populations.
When selecting DNA markers on genetic maps, or later in interpreting linkage data to these markers, it is important to bear in mind how these reference genetic maps were constructed. A genetic map only displays the relative genetic distances between the particularly set of markers that were analysed in a given mapping population. Hence, a given marker will typically have different map positions in genetic maps that were constructed independently, or in successive versions of the same map as new markers are incorporated. In order to construct a better reference map, in particular with respect to the relative order of markers, it is essential that all markers are mapped in the same population. Even with the development of PCR-based markers which require at smaller amount of genomic DNA than RFLP markers, only a limited number of markers and phenotypic traits can be mapped in a given F2 population. This provided the impetus for the generation of populations of recombinant inbred (RI) lines for mapping.
To construct an RI population, individual F2 plants are selfed, and for each F3 family a single F3 plant is selected at random and allowed to self. This process, called single-seed descent, is repeated to the F7. At each generation, the average level of heterozygocity is reduced by 50%. Hence, F7 lines are over 98% homozygous. These RI lines thus constitute permanent mapping populations because they are near-homozygous and can therefore be multiplied indefinitely, enabling multiple laboratories to use the same mapping population.