Normalization of genomic DNA using duplex-specific nuclease
Whole genome shotgun sequencing (WGS) is a potent method for the study of reference sequences in genomes. It generates several sequence data, which result in overlapping sequences eventually. The aligning DNA sequences achieved overlapping sequence reads assembly into contigs, which could read through the computer program. Due to the presence of redundant repetitive sequences in small genomes, the WGS method is not applicable1 (cited in 1). Several methods have proposed to eradicate redundancy in plant genomes which depends on the hypermethylation tendency of repetitive sequences. The use of enzymes or to establish a genomic library could modify the genome, but it is restricted to limited plant genomes 2-4 (cited in 1). Another method proposed in this article called ‘high-C0t DNA analysis’ which is based on DNA renaturation kinetics. In this technique, genomic DNA is denatured and reannealing slowly and then, hydroxyapatite chromatography used for separation of dsDNA. With the help of detailed knowledge of DNA reassociation kinetics and proficiency in spectrophotometry, high-C0t DNA analysis can be applied to any genome5-7 (cited in 1).
Shagina and others, 2010 has discovered the application of duplex-specific nuclease (DSN) normalization technology for eukaryotic genomic DNA. It is a simple and effective method, based on hybridization kinetics excluding physical separation of ssDNA and dsDNA both. After re-association, denatured dsDNA contained repetitive sequence which hydrolyzed by DSN and PCR used for amplification of ssDNA which contain low copy molecules8, 9(cited in 1). DSN enzyme has isolated from the Kamchatka crab which is thermostable and specific to dsDNA10...
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... preparation of normalized cDNA libraries enriched with full-length sequences. Bioorganic Khim. 31:170-177.
10Shagin DA, Rebrikov DV, Kozhemyako VB, Altshuler IM, Shcheglov, Zhulidov PA, Bogdanova EA, Staroverov DB. (2002). A novel method for SNP detection using a new duplex-specific nuclease from crab hepatopancreas. Genome Res. 12:1935-1942.
11Rodrigue S, Malmstrom RR, Berlin AM, Birren BW, Henn MR, and Chisholm SW. (2009). Whole genome amplification and de novo assembly of single bacterial cells. PLoS One 4:e6864.
12Cheung F, Haas BJ, Goldberg SM, May GD, Xiao Y, and Town CD. (2006). Sequencing Medicago truncatula expressed sequenced tags using 454 Life Sciences technology. BMC Genomics 7:272.
13Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K. (2001). Initial sequencing and analysis of the human genome. Nature 409:860-921.
The aim of this experiment was to isolate cDNA molecule CIH-1 (Colletotrichum lindemuthianum CIH1 gene) that is contained in vector pBK-CMV and transfer it into cloning vector pUC19. This was attempted by conducting a restriction digest of vectors pUC19 and pBk-CMV containing CIH-1, using restriction endonucleases Xba1 and EcoR1 and the characterization of recombinant plasmids.
al. (1994) explain that a complementary DNA for GFP produces a fluorescent product when expressed in E. coli cells as the expression of GFP can be used to monitor gene expression and protein localization in living things. In this experiment, the heat shock method will be used to deliver a vector (plasmid) of GFP to transform and grow E. coli bacteria. Four plates containing Luria Bertani (LB) broth and either –pGLO or +pGLO will have E. coli bacteria added to it. The plate containing –pGLO (no pGLO) and LB will show growth as ampicillin will be present killing bacteria but no glowing because no arabinose will be present for glowing to be activated, the same result will be seen in the plate containing +pGLO, LB and ampicillin.
Glase, Jon C. A Study of Gene Linkage and Mapping Using Tetrad Analysis in the Fungus
small sample in need of identification, Polymerase Chain Reactions are used to multiply the DNA
In 1990, the first great stride of genetics took place. This was called the Human Genome Project, a large-scale operation that was designed to understand the human genome (genetic structure). Since its commencement, there have been many leaps and bounds that have taken place. For certain genetic issues that we once knew nothing about, we no...
For the original analysis, the corrected pairwise distance will be calculated using the Jukes–Cantor and the Maximum Composite Likelihood Model. The Jukes–Cantor model assumes that the rate of nucleotide substitution or all nucleotides (C, A, T and G) are equal, that nucleotide frequencies are equal, that there is an equal rate of substitution among sites, and does not correct for the lower rate of transversion substitutes in comparison to transitional substitutions (Jukes and Cantor, 1969). The Maximum Composite Likelihood takes into account the phylogenic relationship between sequences, using the sum of the log likelihoods of the bases as the composite likelihood. Both pair wise distances and substitution parameters are estimated using the Maximum Composite Likelihood (Tamura et al. 2004). Both models should yield different maximum sequence divergence and average divergence that can then be compared to the original paper. With sequence divergence data, the temporal origin of the genus can be identified. The two alternate models to the Kimura-2 parameter will be analyzed to discuss which methods yield results closest to the expected time origin of the genus
Tettelin, H., Nelson, K. E., Paulsen, I. T., Eisen, J. A., Read, T. D., Peterson, S., et al. (2001). Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science. Retrieved from http://www.sciencemag.org/content/293/5529/498.short (Accessed December 12, 2013).
Ridley, M. (1999). Genome: The Autobiography of a Species in 23 Chapters. New York: HarperCollins.
"Fluorescence in Situ Hybridization (FISH) Fact Sheet." National Human Genome Research Institute. 15 Nov. 2007. National Institutes of Health. .
The concept of DNA testing has expanded throughout the last several decades, and attention needs to be paid to the methods and implications of storing and using the samples. The human genome is a complex structure comprised of billions of base pairs. Only 0.1% of DNA makes up all of the differences in humans’ physical appearance (Pattock, 2011, p.855). Each person has about one hundred trillion cells, all of which contain chromosomes that make up an individual’s genome.
Then the sequence was loaded into Velvet where it was trimmed to the desired k-mer length for alignment and contig formation. Mitos and MEGA alignment Explorer were also used in order to get the DNA sequence to a
Genomic sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It includes any method or technology that is used to determine the order of the four DNA bases – thiamine, adenine, guanine, and cytosine– in the strand of DNA (NHGRI, 2011). In each organism, these bases are arranged in a unique and specific sequence, and it is this sequence that is the genetic code of the organism. Genomic sequencing has had an impact on nearly every field of biological research including human genetics and genomics, plants and agriculture, microbes, medicine, viruses and infectious diseases, environmental genetics and evolutionary biology. By first examining the development of gene sequencing technology we will be able to view its role in evolutionary biology, its contribution to phylogenetics, and how it has changed our understanding of the biological tree of life.
Type I markers serve as a bridge for comparison and transfer of genomic information from a map rich species into a relatively map-poor species [11]. Sequence conservation within genes are high, allowing type I markers to serve as anchor points for genomic segments to be compared among species [13]. For instance, if 15 genes are located between type I markers A and B in Zebrafish, it is likely that the majority of the 15 genes also reside between markers A and B in Catfish, even though the exact number of genes, gene order and orientation are not necessarily identical [13].
Bacteria are single celled microbes. Bacteria reproduce by binary fission. In this process, the bacterium, which is a single cell, divides into two identical daughter cells. Binary
The scientific and medical progress of DNA as been emense, from involving the identification of our genes that trigger major diseases or the creation and manufacture of drugs to treat these diseases. DNA has many significant uses to society, health and culture of today. One important area of DNA research is that used for genetic and medical research. Our abi...