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Showing posts with label Genetic Engineering. Show all posts
Showing posts with label Genetic Engineering. Show all posts

Wednesday, 5 September 2007

Large scale gene transfer between organisms

Large scale gene transfer between single-celled and multicellular organisms reported

Wolbachia has on some occasions inserted almost its entire genome into species that it infects reported scientists at the J. Craig Venter Institute and the University of Rochester. This is the first example of large-scale horizontal gene transfer between single-celled and multicellular organisms. Although horizontal gene transmission is common among single-celled organisms, it is rare among multicellular organisms, and large scale transfer like that of an entire genome had previously not been suspected.

The scientists found that in addition to Wolbachia engaging in almost complete genome transfer into Drosophila ananassae, it also had made significant transfer in 3 other insects species and 4 species of nematodes. The researchers found candidate species by scanning genetic databases for sequences found in Wolbachia. The scientists also found that these added sections were conserved by reproduction; that is the added sections stayed in the genomes after multiple generations. Moreover, there is evidence that suggests that the segments of Wolbachia's genome increased the reproductive fitness of the insect species.

The transfers likely occurred during attempts at DNA repair in which the repair mechanisms incorporated Wolbachia DNA (available since the cells were infected with Wolbachia) into the genomes. These results could have major implications for understanding of evolution. The research also has implications for various forms of sequencing research, since when sequencing species, bacteria sequences are frequently ignored as they are generally assumed to be contaminants rather than good data.

Wolbachia, a genus of bacteria that normally infects anthropods, especially insects, is already known for its odd behavior that can affect species in strange ways. For example, Wolbachia has been shown to be correlated with fast evolution among species it infects and is suspected for being responsible for a variety of speciation events as a side affect of Wolbachia creating reproductive barriers. Wolbachia by some estimates infects more than half of all anthropods and is already thought to play a major role in the evolution and speciation of many invertebrates.

Since Wolbachia can generally only reproduce through females, it has adopted a number of strategies that treat males and females of species differently that can result in reproductive barriers. These strategies include killing of males, forced parthenogenesis, and preventing infected males from reproducing with uninfected females.

WikiNews September 4, 2007

Sunday, 24 June 2007

RT-PCR Protocols ebook

RT-PCR Protocols (Methods in Molecular Biology)

This RT-PCR protocol book describes the detail of novel, useful, and
interesting RT-PCR applications.

protocols available for

highly sensitive detection and quantification of gene expression, the in situ localization of gene,
expression in tissue, and the cloning of genes,

as well as for analyzing T-cell clones and the differential expression of genes,

including laser-capture microdissection (LCM),

real-time and quantitative PCR,

microarray technology, cDNA cloning.

Book Info:
Published in 2002
Published by Humana press
Author Joseph OConnell
ISBN 0896038750  Size 4.30MB

Link

Saturday, 16 June 2007

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology
ISBN: 047150338X
Author: Roger Brent / Robert E. Kingston / J. G. Seidman / Kevin Struhl / Frederick M. Ausubel / Virginia Benson Chanda / David D. Moore / J.G. Seidman / F.M. Ausubel
Publisher: John Wiley & Sons Inc
Edition: ringbou edition (December 4, 2003)
ISBN: 047150338X
Ring-bound: 1600 pages
URL: /http://www.amazon.com/exec/obidos/redirect?tag=songstech-20&path=ASIN%2F047150338X
Summary:

Current Protocols in Molecular Biology, the first Current Protocols publication, remains the international standard by which all other lab manuals are judged. Basic methods for DNA preparation and isolation, library screening, and sequencing have been joined by more advanced procedures detailing DNA-protein interactions, yeast manipulation, and phosphorylation analyses. From basics to the cutting edge, CPMB is the only resource you need for successful experiments.

Link

Friday, 15 June 2007

MicroRNA Protocols (Methods in Molecular Biology)

MicroRNA Protocols (Methods in Molecular Biology)

  • Publisher: Humana Press
  • Number Of Pages: 272
  • Publication Date: 2006-04-01
  • Sales Rank: 1248190
  • ISBN / ASIN: 1588295818
  • EAN: 9781588295811
  • Binding: Hardcover
  • Manufacturer: Humana Press
  • Studio: Humana Press
  • Book Description:

    MicroRNA Protocols provides diverse, novel, and useful descriptions of miRNAs in several species, including plants, worms, flies, fish, chicks, mice, and humans. These include some useful adaptations and applications that could be relevant to the wider research community who are already familiar with the identification of miRNAs. This volume will stimulate the reader to explore diverse ways to understanding the mechanism in which miRNAs facilitate the molecular aspects of the biomedical research.

  • Link

    pass:lekar

    Thursday, 14 June 2007

    PCR Protocols-Methods in Molecular Biology EBook

    PCR Protocols: Current Methods and Applications (Methods in Molecular Biology)

  • Publisher: Humana Press
  • Number Of Pages: 392
  • Publication Date: 1993-01
  • Sales Rank: 914567
  • ISBN / ASIN: 0896032442
  • EAN: 9780896032446
  • Binding: Spiral-bound
  • Manufacturer: Humana Press
  • Studio: Humana Press

    Link

  • Pass: econiches

    Wednesday, 13 June 2007

    Standard PCR Protocol

    Standard PCR Protocols

    This protocol is link to Molecular Biology Techniques Manual
    Third Edition
    Edited by:
    Vernon E Coyne, M Diane James, Sharon J Reid and Edward P Rybicki

    Read Full Protocol

    Overlap Extension PCR

    Overlap Extension PCR is used to create long DNA fragments from short ones.

    or used for Engineering the replication of target DNA through cloning, or changing its genetic code through mutations

    PCR amplify the necessary fragments, using polymerase enzyme. They should have about 15-25 bp overlaps. Use oligo Tm calculators to figure out their annealing temp.
    Clean up or gel extract the correct size band. Use cleaned up fragments as "template". Unlike normal PCR, about 1/2 to 3/4 volume of the extension reaction should be template.
    Use proofreading enzyme for extension. Run 3 reactions of 10,15 and 30 PCR cycles without end primers. (Template extension step) Add end primers, then continue cycling for another 15-20 rounds. Gel extract the correct fragment. Clone into a your desired vector.

    check out the latest Nature Methods Protocol
    http://www.nature.com/nmeth/journal/v4/n5/pdf/nmeth0507-455.pdf

    Primer Sets and PCR Manual


    This 24-page brochure details each step of the Polymerase Chain Reaction process with technical information for basic PCR techniques, methods, applications and polymerase choices. You will want to keep this new booklet close at hand because it also includes FAQs, references and a troubleshooting guide.


    Friday, 8 June 2007

    High-Yield Biostatistics (High-Yield Series)-E-Book

    Synopsis
    Part of the successful High-Yield "TM" Series, High-Yield "TM" Biostatistics, Second Edition explains concepts, provides examples, and covers the complete range of biostatistics material that can be expected to appear on the USMLE Step 1. New to this edition are references to evidence-based medicine, and information updated to reflect changes in the current USMLE examinations.
    Table of Contents
    Descriptive statistics - Inferential statistics - Hypothesis testing - Correlational techniques - Research methods - Statistics in epidemiology - Statistics in medical decision making - Ultra-high-yield review

    Download

    Handbook of Statistics in Clinical Oncology-E-Book

    Handbook of Statistics in Clinical Oncology

    Chapman & Hall/CRC
    ISBN: 0824723392
    640pages
    2006-
    Pdf-English
    size:17,42 Mb

    Book Description
    A compendium of cutting-edge statistical approaches to solving problems in clinical oncology, this book focuses on clinical trials in phases I, II, and III, proteomic and genomic studies, complementary outcomes and exploratory methods. Cancer Forum called the first edition a "good reference book for statisticians who will be designing and analyzing cancer trials." The second edition includes over 1000 references, more than forty world-renowned contributors, and 300 equations, tables, and drawings. Completely revised while keeping the features that made the first edition a bestseller, this is the best single source for up-to-date statistical approaches to research in clinical medicine.
    Features:
    Provides a comprehensive discussion of sample size
    Explores analytical problems generated by controlling treatment costs and maintaining quality of life
    Demonstrates the breadth and depth of current activity in the field of survival analysis
    Includes recommendations and pointers for free software that allows you to implement programs
    Sets the limits on what can and cannot be concluded from single and multiple clinical trials
    Contents
    Phase I Trials
    Phase II Trials
    Phase III Trials
    Exploratory Analysis and Prognostic Factors
    High-Throughput Data and Bioinformatics
    Interpreting Clinical Trials

    Download

    Statistical Methods in Bioinformatics: An Introduction-E-Book

    • Hardcover: 588 pages
    • Publisher: Springer; 2 edition (September 30, 2005)
    • Language: English
    • ISBN-10: 0387400826

    Advances in computers and biotechnology have had a profound impact on biomedical research, and as a result complex data sets can now be generated to address extremely complex biological questions. Correspondingly, advances in the statistical methods necessary to analyze such data are following closely behind the advances in data generation methods. The statistical methods required by bioinformatics present many new and difficult problems for the research community.

    This book provides an introduction to some of these new methods. The main biological topics treated include sequence analysis, BLAST, microarray analysis, gene finding, and the analysis of evolutionary processes. The main statistical techniques covered include hypothesis testing and estimation, Poisson processes, Markov models and Hidden Markov models, and multiple testing methods.

    The second edition features new chapters on microarray analysis and on statistical inference, including a discussion of ANOVA, and discussions of the statistical theory of motifs and methods based on the hypergeometric distribution. Much material has been clarified and reorganized.

    The book is written so as to appeal to biologists and computer scientists who wish to know more about the statistical methods of the field, as well as to trained statisticians who wish to become involved with bioinformatics. The earlier chapters introduce the concepts of probability and statistics at an elementary level, but with an emphasis on material relevant to later chapters and often not covered in standard introductory texts. Later chapters should be immediately accessible to the trained statistician. Sufficient mathematical background consists of introductory courses in calculus and linear algebra. The basic biological concepts that are used are explained, or can be understood from the context, and standard mathematical concepts are summarized in an Appendix. Problems are provided at the end of each chapter allowing the reader to develop aspects of the theory outlined in the main text.

    Warren J. Ewens holds the Christopher H. Brown Distinguished Professorship at the University of Pennsylvania. He is the author of two books, Population Genetics and Mathematical Population Genetics. He is a senior editor of Annals of Human Genetics and has served on the editorial boards of Theoretical Population Biology, GENETICS, Proceedings of the Royal Society B and SIAM Journal in Mathematical Biology. He is a fellow of the Royal Society and the Australian Academy of Science.

    Gregory R. Grant is a senior bioinformatics researcher in the University of Pennsylvania Computational Biology and Informatics Laboratory. He obtained his Ph.D. in number theory from the University of Maryland in 1995 and his Masters in Computer Science from the University of Pennsylvania in 1999.

    Comments on the First Edition. "This book would be an ideal text for a postgraduate course.[and] is equally well suited to individual study.. I would recommend the book highly" (Biometrics). "Ewens and Grant have given us a very welcome introduction to what is behind those pretty [graphical user] interfaces" (Naturwissenschaften.). "The authors do an excellent job of presenting the essence of the material without getting bogged down in mathematical details" (Journal. American Staistical. Association). "The authors have restructured classical material to a great extent and the new organization of the different topics is one of the outstanding services of the book" (Metrika).

    Download

    Wednesday, 6 June 2007

    The Cell - A Molecular Approach-E Book, The Cell - A Molecular Approach Cooper, Geoffrey M. Sunderland (MA): Sinauer Associates, Inc. ; c2000

     

     

     

     

    The Cell - A Molecular Approach

    Cooper, Geoffrey M.

    Sunderland (MA): Sinauer Associates, Inc. ; c2000

    Read This Book Full Version

    C. elegans-Ebook, Riddle, Donald L.; Blumenthal, Thomas; Meyer, Barbara

    C. elegans II

    Riddle, Donald L.; Blumenthal, Thomas; Meyer, Barbara J.; Priess, James R., editors.

    Plainview (NY): Cold Spring Harbor Laboratory Press ; c1997

    Read This Book Full

    TA cloning or Subcloning of PCR Products Protocol

    TA Subcloning of PCR Products

    This procedure is adapted from D. Marchuk, M. Drumm, A. Saulino, and F.S. Collins Nuc. Acids. Res. (1991) 19:1154.
    CONSTRUCTION OF T-VECTOR
    1. suspend 10ug pUC 19 in:
      • 4.0ul 10X reaction buffer (we use Bo. Mann. buffer A)
      • 2.0ul (20U) Sma I
      • X ul dwater to a total vol. of 40ul
        Incubate at 30 (not 37) degrees for 1 hour. This is easier if done in a 0.4ml tube in a thermal cycler.
    2. Heat to 70 degrees for 15 min. to kill the enzyme
    3. Bring to 100ul w/ water (add 60ul).
    4. Extract w/ phenol, phenol/chloroform and then chloroform.
    5. add 9ul 3M sodium acetate.
    6. ppt. in ETOH, wash with 70% ETOH (be careful with the pellet!).
    7. Dry in spin vac at room temp (do not use heater!).
    ********************T-TAILING THE VECTOR******************
    At this point, it is assumed that there has been 80% recovery of the cut plasmid DNA.
    1. Resuspend the plasmid DNA in 63ul water (conc approx. 130ng/ul)
    2. To the resuspended plasmid add:
      • 10ul 10X PCR buffer (standard cetus stuff, no MgCl)
      • 20ul 10mM dTTP [2mM final]
      • 6ul 25mM MgCl2 [1.5mM final]
      • 1ul Taq polymerase (Cetus amplitaq 5U/ul)
      • ______
      • 100ul total volume.
    3. Incubate for 3 hours at 70 degrees C.
    4. Extract with Phenol, Phenol/chloroform, chloroform.
    5. Extract twice with ether (so I'm paranoid!)
    6. add 75ul 2M **ammonium** acetate (assuming 75ul recovery from extractions).
    7. Add 150ul isopropanol. Spin 20mins in microfuge at full speed at 4 degrees.
    8. Wash with 70% ETOH 9 Dry pellet in spin vac and store at -20 degrees until use.
    TREATMENT THE PCR PRODUCTS
    " If you can see it, you can clone it".
    1. Add an equal volume of chloroform (*NO* IAA) to the PCR reaction and spin 1-2 minutes in microfuge at RT.
    2. Remove the oil which is now on the ****BOTTOM***.
    3. Spin again for two minutes and remove the last little bit of oil from the bottom. You will know when you have gotten it all when you see the interface in the pipette tip. It is important that all the oil be removed otherwise subsequent procedures will be very difficult.
    4. Add 100ul 4M ammonium acetate, vortex, and then add 200ul isopropanol.
    5. Centrifuge 20min at 4 degrees, wash in 70% ETOH.
    6. Dry in speed vac.
    7. Resuspend the DNA in 8-10ul TE, add loading buffer and load onto a 4% Nusieve (TAE) agarose gel. Run until the desired band is well separated. The more DNA in the band, the easier it is to subclone.
    8. Cut out the band. Minimize the exposure of the gel (and you!) to short wave UV
    LIGATION OF PCR PRODUCTS TO T-VECTOR
    1. Heat the gel containing the PCR fragment to 65C for 10 minutes, place in a 37C water bath or block and add to a separate tube (also at 37C):
      • 10 ul gel
      • 4ul 5X ligase buffer (commercial buffer that comes with BRL T4 ligase)
      • 4ul water
      • 1ul vector (25-50ng)
      • 1ul ligase
      • Incubate at 12C overnight.
    2. Heat the mixture to 68 degrees for 5 minutes and add 100ul water.
    3. Extract with phenol, phenol/chloroform, and chloroform. These steps are to remove the agarose.
    4. Add 10ul 3M NaAcetate and precipitate with ethanol.
    5. Wash the pellet in 70% ETOH, dry in the speed-vac. Resuspend in 5ul of water just prior to transformation.
    *Transformation - We usually use electroporation into XL-1 blue cells. You need cells that can achieve at least 1 x 10^7 transformants per ug of DNA if a CaCl based protocol is used.

    *Storage: The T-vector should be stored at -20C at all times. When stored in dry form, the T-overhangs will last longer (I don't know how long yet). In solution, it lasts at least a couple of weeks at - 20C.

    *Enzymes - The batch of SmaI that is used is particularly critical. Some are contaminated with an endonuclease that removes a few bases from the cloning site. The batch of smaI should be checked before it is used to cut vector for cloning purposes. If bluescript is used, EcoRV can be substituted for sma I.

    txpljfg@uabcvsr.cvsr.uab.edu

    Cloning of Blunt-end PCR Fragments Protocol

    Direct Cloning of Blunt-end PCR Fragments

    BioTechniques 13:613
    • Phenol extract the PCR product
    • Ethanol precipitate
    • Treat for 1hr at 37C with 10 units of T4 DNA polynucleotide kinase and 10 units T4 DNA polymerase I(NEB) in a 100ul reaction volume containing 50mM Tris-HCl pH7.5, 10mM MgCl2, 1mM DTT, 50 ug/ml BSA, 1mM ATP, 200uM each dNTP.
    I run the entire thing out on a 1.3% agarose TAE gel, cut a trough in front of the band, pour in some 0.7% LMP agarose(BRL), run the product into it and excise.

    The PCR product in the LMP can be used for ligations directly, without purification. The ligations take place at room temp on the benchtop. I prepare the vector with minimal digestion (~2hr) then treat it with shrimp alkaline phosphatase(USB). I usually prepare a stock of this vector to have on hand, so I know it is good and will have a low background. You may also want to try using an EcoRV cut vector instead of a Sma cut vector.

    • Remove the oil with Diethylether.
    • To 40 ul of the PCR reaction add 50 ul of H2O. Add 10 U T4 PNK, 10 U klenow, ATP (to 1 mM) and some more dNTPs (usually 4 ul of 1.25 mM...whatever) and icubate at 37 oC for 30 min.
    • Phenol/chloroform extract
    • chloroform/IAA extrac
    • EtOH precipitate
    you can then just "shotgun" clone this DNA. However, if you have several non-specific bands then you may want to gel purify the fragment first.

    One point to note....

    We have given up blunt end cloning into Sma I sites where possible. Several people have reported problems with Sma I cut DNA. By choice we clone into EcoRV sites.

    Sunday, 3 June 2007

    What is DNA REPAIR?

    simple again.. you damage ur DNA by smoking.. n repair it by urself...learn how..

    What is transcription?

    Simple... formation DNA to RNA is transcription.. learn more.. easy n nice.. cool video

    What is DNA MUTATION?

    Simple, nice n easy way to learn how DNA mutation works... enjoy

    What is HIV?

    Learn What is HIV and How it cause infection and how our immune system works.

    Science Protocols