Volume 11, Issue 3, 2011 July-September

Volume 11, No 3 Pages:
2011 July-September Articles: 10

Random amplified polymorphic DNA (RAPD) markers and its applications

In the last decade, the random amplified polymorphic DNA (RAPD) technique based on the polymerase chain reaction (PCR) has been one of the most commonly used molecular techniques to develop DNA markers. RAPD is a modification of the PCR in which a single, short and arbitrary oligonucleotide primer, able to anneal and prime at multiple locations throughout the genome, can produce a spectrum of amplification products that are characteristics of the template DNA. RAPD markers have found a wide range of applications in gene mapping, population genetics, molecular evolutionary genetics, and plant and animal breeding. This is mainly due to the speed, cost and efficiency of the technique to generate large numbers of markers in a short period compared with previous methods. Therefore, RAPD technique can be performed in a moderate laboratory for most of its applications. It also has the advantage that no prior knowledge of the genome under research is necessary.

Protein 3D structure determination using homology modeling and structure analysis

The protein sequence of aspartate aminotransferase of pig was retrieved from the Swiss-Prot database. The appropriate template for homology modeling was determined using Blastp. 3D structures were determined by homology modeling softwares such as Swiss Model and EsyPred3D which passed quality test by ProQ software and set for further analysis. The pockets determined by CASTp server for the predicted structures showed a significant difference in the pocket area and volume, which were due to structural deviation between the residues 30-40 found in the 3d-ss software. Both the structures were analyzed using ProFunc tool which showed different functions as they had different structures and active sites. Thus the structure plays a vital role in determining its function.

Primer designing for PCR

Design your PCR primers to be 18-30 oligonucleotides in length. The longer end of this range allows higher specificity and gives you space to add restriction enzyme sites to the primer end for cloning. Make sure the melting temperatures (Tm) of the primers used are not more than 5°C different from each other. You can calculate Tm with this formula: Tm = 4(G + C) + 2(A + T)°C. Aim for a Tm between 52 and 58°C for each primer over the region of hybridization. Use an annealing temperature (Ta) of 3-5°C lower than the Tm. The GC content of each primer should be in the range of 40-60% for optimum PCR efficiency. Try to have uniform distribution of G and C nucleotides, as clusters of G’s or C’s can cause non-specific priming. Avoid long runs of the same nucleotide. Check that primers are not self-complementary or complementary to the other primer in the reaction mixture, as this will encourage formation of hairpins and primer dimers and will compete with the template for the use of primer and reagent. If you can, make the 3′end terminate in C or A, as the 3′is the end which extends and neither the C nor A nucleotide wobbles. This will increase the specificity. You can avoid mispriming by making the 3′end slightly AT rich. Use the right software. Using the right software is a great way to automate these steps and minimize errors, especially when you have to design primers for many sequences.

Biological databases with emphasis on biodiversity and conservation

Bioinformatics implements the use of biology, computational mathematics, computer science and information technology. Through the combination of these methods, scientists are able to store and compare the information from all kinds of species and how they evolve. The complex and voluminous data of biodiversity can be digitalised for easy accession, analysis and interpretation. It makes easy survey, documentation and measurement of biodiversity data. The data bases, management and their applications in general as well as in relation to biodiversity and conservation are discussed.

Morphological and molecular studies of six Junonia species of butterflies using RAPD-PCR technique

The genomic DNA of six species of butterflies (Junonia atlites, J. iphita, J. hierta, J. orithiya J. lemonias and J. almanac), family Nymphalidae, sub-family Nymphalinae, were used for RAPD-PCR analysis using 15 random primers to study the genetic similarity and diversity. A total of 437 bands were scored, of which 357 were polymorphic and the average percent polymorphism was 82.70%. Dendogram constructed using the UPGMA of NTSYS spc2.2 software divided the Junonia species into two clades. There is a difference in the branching pattern between the morphological and molecular data, which signifies the need for using molecular tools for taxonomic classification as well as in understanding the evolutionary relationship.

DNA finger printing of Bacillus thuringiensis based on repetitive DNA sequences using ERIC-PCR

To assess the distribution and evolutionary conservation of distinct prokaryotic repetitive elements, consensus oligonucleotides were processed in polymerase chain reaction (PCR) amplification with genomic DNA from different Bacillus thuringiensis (Bt) strains from different parts of Mizoram, India. Oligonucleotides matching enterobacterial repetitive intergenic consensus (ERIC) sequences were synthesized and tested as opposing PCR primers and produced clearly resolvable bands by gel electrophoresis, which provided unambiguous DNA fingerprints of the different Bt strains. After analysing with NTYSYS, DARwin and POWERMARKER, a dendogram was constructed, which revealed that the Bt strains were divided into three main clusters. Widespread distribution of the repetitive DNA elements enabled rapid identification of these Bt strains.

Evaluation of the random amplified polymorphic DNA (RAPD) assay for the detection of DNA damage in mosquito larvae treated with plant extracts

The random amplified polymorphic DNA (RAPD) assay was used to assess the level of DNA damage in various exposed and unexposed Culex quinquefasciatus larvae to acetone and chloroform extracts of Curcuma longa and Melia azedarach at different concentrations (6.25, 12.5 and 25 ppm). This is the first report of an analysis of genomic alterations in plant extracts-treated mosquito larvae using RAPD-PCR fingerprinting. In comparison to the control larvae, larvae treated with the plant extracts caused greater changes in the RAPD patterns. DNA strand breakage was more in the larvae of C. quinquefasciatus.

RAPD-PCR characterization of insecticide resistant and susceptible Anopheles species

Anopheles barbirostris is the major vector of Timor filaria, and A. jamesii is of Bancroftian filaria in Srilanka, and is a suspected vector of malaria as it can support the sporogenic cycle of Plasmodium vivax. Deltamethrin screening revealed that A. jamesii was susceptible to deltamethrin (LC50 = 0.0025 ppm; LT50 = 11.38 min), while A. barbirostris was resistant (LC50 = 3.802 ppm; LT50 = 20.28 min). Genomic DNA isolated from the two species were used for characterisation of the insecticide resistance and susceptibility using Random Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR). 15 random primers produced 59 bands in A. barbirostris and 31 bands in A. jamesii. Out of these, 58 bands were polymorphic between the two species. The genetic distance calculated using FREE TREE software indicated 0.91549 (Nei and Li method) and 0.83824 (Jaccard method). The matrices for dissimilarity (1.358) and similarity (0.150) between the two species calculated using NTSYSpc 2.2 showed polymorphism of 88.9 %. Genetic variance between A. barbirostris and A. jamesi is probably the reason for the former to be resistant, while the latter is susceptible to deltamethrin.

Genetic variation within two cryptic species of Cirrochroa (Heliconiinae: Lepidoptera) by RAPD-PCR technique

The morphological characters of Cirrochroa aoris (Large Yeoman) and C. tyche (Common Yeoman) are very similar making identification confusing and difficult. The genomic DNA of the two species was subjected to RAPD-PCR analysis with six decamer oligonucleotides, i.e. MA5, MA6, MA8, OPB12, OPT4 and OPT5. All of them produced discrete bands of various lengths revealing genetic variations as well as similarities between the two species. A total of 50 RAPD bands were generated with 45 polymorphic bands. The percentage polymorphism was 90.58% and all the similarity coefficients between the species were less than 0.2. Results showed a high genetic variation between the two cryptic species. Some species specific bands were obtained with these primers which can be considered as diagnostic bands. All the primers also produced species specific bands.

PCR pathotyping of native Bacillus thuringiensis from Mizoram, India

Bacillus thuringiensis is a ubiquitous, gram-positive and spore-forming bacterium. During sporulation, it produces intracellular crystal (cry) proteins, which are toxic to insects. The genetic diversity of B. thuringiensis strains shows regional differences. Thus, each habitat may contain novel strains with new insecticide. The aim of this study was to isolate B. thuringiensis strains from different environments of Mizoram, India, and to identify the cry gene content of the isolates using PCR. The universal primers specific to cry1, cry2, cry3, cry4 and cry9 genes were used to detect the type of cry gene carried by each environmental isolate. Altogether, a total of 42 cry genes were detected out of which 12 were cry1, 5 were cry2, 3 were cry3, 18 were cry4 and 22 were cry9 out of 45 selected strains.