In this paper authors discussed of using single molecule PCR (smPCR) to address the bypass-related PCR articificial mutation amplification. Mitochondria DNA mutation detection by PCR is troublesome and this technique paper discussed in detail some of the aspects of somatic mtDNA mutation detection.

Abstract:

Single molecule PCR in mtDNA mutational analysis: Genuine mutations vs. damage bypass-derived artifacts. Methods. 2008 Oct 24.
Kraytsberg Y, Nicholas A, Caro P, Khrapko K.

The area of somatic mtDNA mutation measurement is in a crisis because the methods used to quantify mtDNA mutations produce results varying by multiple orders of magnitude. The reason for these discrepancies is not clear, but given that most methods involve PCR, the prime suspect is PCR artifacts (e.g. spontaneous errors by the DNA polymerases used). In addition to simple misincorporation, another important source of artificial mutations is the conversion of chemically modified (e.g. damaged) nucleotides into mutations when bypassed by a thermostable DNA polymerase. This source is particularly worrisome because in a mutational spectrum obtained it is quite difficult to estimate the fraction of mutations that resulted from conversion of from DNA damage of the template. Here, we argue that single molecule PCR (smPCR) is uniquely positioned to account for these bypass-related artificial mutations and discuss the methodology involved in employing this technique.

I got positive results from southern hybridization for my gene while screening genomic library. The library was made using Novagen’s lambda bluestar vector arms. The selected plaque was positive till third round of screening, however it gave blue colonies on excision from lambda blue star vector. The sequencing results also showed the presence of LacZ gene and absence of my insert. Could anyone help me or suggest anything. The probe is made through PCR on PCRII topo vector containing the fragment.

Cheers
asifa

the specificity of the interaction in base pairing during DNA or RNA synthesis enables production of specific DNA probes

Bioinformatics and Genomics play an increasingly important and transformative role in pharmaceuticals, medical, biotechnology, microbiology, agriculture, life sciences and society. Bioinformatics is the use of computers to retrieve, process, analyze and simulate molecular biology information. The mapping of the human genome has revealed 35,000 or so genes, which might code for more than one protein, resulting in 100,000 proteins for the humans alone. Since proteins are attractive targets for developing drugs, researchers and scientist’s efforts are now underway to map sequences and assign functions to many novel proteins. It promises to revolutionize the process of drug discovery and development.
This book provides a broad, application-oriented overview of bioinformatics and biotechnology. In addition, the state-of-the-art in bioinformatics is evaluated equally from a global view by introducing real application scenarios such as genome projects that require the use of a whole set of bioinformatics tools. This appears to be an excellent textbook for students, researchers, scientists and bioinformaticians. This book is a very valuable and important resource for bringing bioinformatics into the work practice of geneticists. Also this is essential reading for all those engaged in genetic and new drug development research. This book takes the novel approach to cover the methods, sequence and structure analysis of nucleotides, proteins in one volume and form an algorithmic perspective.
Most of the chapters include tables that are designed to propose an orderly use of methods that are discussed in the chapters. There are very few examples of these types of tables and over simplification that may not always justify. We hope that these tables and figures will be useful for the less experienced in this field, but we expect that the more experienced researchers in the field will have other, probably better, way of achieving the same goal.
There are many references to web address and FTP locations where these methods may be applied or program obtained is given in the book. In some cases, as far the commonly used bioinformatics programs, we have provided a grate deal of information about using the programs, tools for analyzing the results. There are many other important bioinformatics tools that are available for sequence analysis. We have tried to cover as many of them.
In writing this book, we found that the amount of information available in the published literature was far more than we could include. We have tried to be through and to cover the most significant problems in the sequence analysis, but there are also many excellent literature that have not been cited for reason of time and space.
This book provides a basic understanding of the theories, associated algorithms, resources, and tools used in functional and structural bioinformatics for genetic research. The reader emerges with the ability to make effective use of protein, DNA, RNA, and complex structures to better understand biological function. Moreover, it draws a clear connection between sequence studies and the rational design of new therapies.
The profound knowledge on bioinformatics presented here not only enables readers to go beyond a mere push-button approach to using bioinformatics software and interpreting the data generated appropriately. It is also essential to assess the potential of today’s bioinformatics software and future challenges. Bioinformatics theory and practices is a timely and much-needed textbook, which provides an accessible and thorough introduction to a subject. Which has become a fundamental part of biological science today. Content of book is helpful for analysis of DNA, RNA and proteins. The aim of the book is to integrate an understanding of the biological background of bioinformatics with development of necessary computing skills. Without describing computer science or sophisticated programming skills in detail, the book supports and encourages the application of the many powerful computational tools of bioinformatics in a way that is both relevant to and stimulating for reader.
The book contains numerous online analysis examples to encourage students, researchers to engage with the subject and with the accompanying web site, to develop a working understanding and appreciation of the power of bioinformatics as a research tool. This book is highly recommended for academic, biotechnology, pharmaceuticals, medical, agriculture, genomics, proteomics, life sciences and for researchers as a theory and practical. Although this book is aimed at professional geneticists, the numerous worked examples would be useful to anyone putting a course together on this topic.
No words are enough to express our feelings, love and indebtedness for the contribution of our friends and family members to outcome this book. Their zeal is the constant source of encouragement for us.

Virendra S. Gomase

Book:

Bioinformatics- Theory and Practice

N.J. Chikhale and Virendra Gomase

• Paperback: 592 Pages
• Publisher: Himalaya Publication House, India; 1 edition (July, 2007)
• Language: English
• ISBN-13: 978-81-8318-831-9

Table Content

1. Introduction of Bioinformatics
2. The Central Dogma of Biology
3. Amino Acid Information
4. Functional Genomics
5. Biological Database & Databanks-I
6. Biological Database & Databanks-II
7. Name & Function of Database
8. Retrieval of Data and Its Description
9. Gene Expression Analysis
10. Microarray & Gene Expression
11. Pairwise Sequence Alignments
12. Fasta & Blast
13. Multiple Sequence Alignments
14. Methods of Gene Prediction
15. RNA Secondary Structure Prediction

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i need to know wat factors can lead to shearing of dna during extraction by phenol-chloroform method?

A new method on quantitative profiling of signaling networks using phosphotyrosine-specific DNA-tagged SH2 domains was reported by Dierck et.al and published on Nature methods (2006,3:737-744). The new proteomic technique, termed OTM, is able to quantify tyrosine phosphorylation states with high sensitivity and specificity.

Quantitative multiplexed profiling of cellular signaling networks using phosphotyrosine-specific DNA-tagged SH2 domains. Dierck K. et.al., Nat Methods. 2006 Sep;3(9):737-44

[Abstract] Deciphering global signaling networks is of great importance for the detailed understanding of cellular signaling processes controlling many important biological functions. Among signaling processes, tyrosine phosphorylation has a central role. At present, adequate techniques for the global characterization of the tyrosine phosphoproteome are lacking, particularly for the analysis of small amounts of protein. By combining the power of PCR amplification with the unique properties of Src homology region 2 (SH2) domains to specifically recognize tyrosine-phosphorylated proteins, we developed a new proteomic approach, termed oligonucleotide-tagged multiplex assay (OTM). For OTM, multiple SH2 domains are labeled by domain-specific oligonucleotide tags, applied as probes to complex protein mixtures in a multiplex reaction and phosphotyrosine-specific interactions are quantified by PCR. Using OTM we reproducibly quantified differential states of tyrosine phosphorylation with high sensitivity and specificity in small amounts of whole cellular extracts as demonstrated for various tumor cell lines and human leukemia samples.

Related methods and protocols:

quantitative real-time PCR
Proteomics protein modification protocol

ChIP-on-chip is a genome-wide location analysis to identify protein-binding site, including promoters, enhancers, and other regulatory elements. Here is a nice ChIP-on-chip technique illustration from Keith Ching at UCSD.

Following is a protocol from nature for genome-wide transcriptional binding site analysis using ChIP-on-chip. The entire method takes 10 days to complete.

ChIP-on-chip protocol for genome-wide analysis of transcription factor binding in Drosophila melanogaster embryos

Thomas Sandmann et.al.
Nature Protocols 1, - 2839 - 2855 (2007)

This protocol describes a method to detect in vivo associations between proteins and DNA in developing Drosophila embryos. It combines formaldehyde crosslinking and immunoprecipitation of protein-bound sequences with genome-wide analysis using microarrays. After crosslinking, nuclei are enriched using differential centrifugation and the chromatin is sheared by sonication. Antibodies specifically recognizing wild-type protein or, alternatively, a genetically encoded epitope tag are used to enrich for specifically bound DNA sequences. After purification and polymerase chain reaction-based amplification, the samples are fluorescently labeled and hybridized to genomic tiling microarrays. This protocol has been successfully used to study different tissue-specific transcription factors, and is generally applicable to in vivo analysis of any DNA-binding proteins in Drosophila embryos. The full protocol, including the collection of embryos and the collection of raw microarray data, can be completed within 10 days.

Dr.Andrew Z.Fire from Stanford University and Dr. Craig C.Mello from University of Massachusetts Medical School shares 2006 Nobel prize in Physiology or Medicine for their achievement of “discovery of RNA interference - gene silencing by double-stranded RNA” announced on October 2, 2006.

Ongoing research programs are listed in their lab homepage:

Fire lab research program

Mello lab at Umass
Dr.Mello’s research program at Howard Hughes Medical Institute

RNAi protocols and troubleshootings

Joining few of online bioscience protocol publishers, Nature launched
online protocol section
in May 2006. As quoted from nature protocols, “Nature Protocols is an
online resource for protocols, including authoritative, peer-reviewed
‘Nature Protocols’ and an interactive ‘Protocols Network’. The two
create a dynamic forum for scientists to upload and comment on
protocols. Protocols Network is currently in Beta phase and further
functionality will be added over the coming weeks.”

Other methods and protocol focused journals include:

NCICB developed this pathway interaction database as part of cancer molecular analysis project. Currently it contains 5,000 interactions, 2200 proteins, 350 complexes and 2300 small molecules derived from 99 KEGG metabolic pathways and 122 BioCarta signaling pathways.

Address: Pathway database