5 Key excerpts on "DNA Hybridisation"

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  eBook - ePub

  Molecular Biology

  • David P. Clark(Author)
  • 2009(Publication Date)
  • Academic Cell

    (Publisher)

  Fig. 21.31 ). Some of the single strands of DNA molecule No. 2 will base pair with the single strands of DNA molecule No. 1 and will stick to the filter. (As discussed above, DNA molecule No. 2 must be labeled by radioactivity, fluorescence or some other way to enable its detection.) The more closely related the two molecules are, the more hybrid molecules will be formed and the higher the proportion of molecule No. 2 that will be bound by the filter. For example, if the DNA for a human gene, such as hemoglobin, was fully melted and bound to a filter, then DNA for the same gene but from different animals could be tested. We might expect gorilla DNA to bind strongly, frog DNA to bind weakly and mouse DNA to be intermediate. [Several variants of nucleic acid hybridization are in use. This version, involving the binding of DNA to DNA is known as Southern blotting—see below.]

  Figure 21.31 Relatedness of DNA by Filter Hybridization (A) DNA No. 1 is denatured and attached to a filter. (B) When DNA No. 2 is added to the filter, some of the DNA strands will hybridize, provided that the sequences are similar enough. If the sequence is identical, all of the single-stranded red DNA should hybridize to strands of green DNA. If the sequences are very different, little or none of the green DNA will hybridize with the red.

  Hybrid double helices may be formed by annealing single strands that are related in sequence.

  Another use for hybridization is in isolating genes for cloning. Suppose we already have the human hemoglobin gene and want to isolate the corresponding gorilla gene. First the human DNA is bound to the filter as before. Then gorilla DNA is cut it into short segments with a suitable restriction enzyme (see Ch. 22 for details). The gorilla DNA is heated to melt it into single strands and poured over the filter. The DNA fragment that carries the gorilla gene for hemoglobin will bind to the human hemoglobin gene and remain stuck to the filter. Other, unrelated genes will not hybridize. This approach allows the isolation of new genes provided a related gene is available for hybridization.

  A wide range of methods based on hybridization is used for analysis in molecular biology. The basic idea in each case is that a known DNA sequence acts as a “probe .” Generally the probe molecule

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  eBook - ePub

  Recombinant DNA Principles and Methodologies

  • James Greene, James Greene(Authors)
  • 2021(Publication Date)
  • CRC Press

    (Publisher)

  8

  Hybridization Probes

  Frank J. Castora Eastern Virginia Medical School, Norfolk, Virginia James J. Greene The Catholic University of America, Washington, D.C.

  I. INTRODUCTION

  Nucleic acid hybridization is the basis for a broad array of methods that allow for the detection and quantitation of nucleic acids. It involves the formation of hydrogen bonds between two complementary strands of nucleic acid resulting in the production of a double-stranded complex. Most applications of hybridization such as Southern and northern analysis (Chapter 7 ) utilize a labeled probe that is hybridized to a target nucleic acid of interest. The probe is nucleic acid that is complementary to the target and is labeled with a molecule or radioisotope that makes it easily detectable.

  In most of these applications, the nucleic acid sample to be analyzed is immobilized on a filter membrane in the single-stranded state. If DNA is the sample, it is first denatured into its component single strands before final immobilization on the membrane. Incubation of the filter with the probe results in hybridization of some of the probe to the filter if the sample contains the specific target nucleic acid. The visualization of the signal that is associated with the radioisotope or another type of label provides information regarding the presence, location, and amount of the target nucleic acid of interest.

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  eBook - ePub

  Medical Biochemistry

  • Antonio Blanco, Gustavo Blanco(Authors)
  • 2017(Publication Date)
  • Academic Press

    (Publisher)

  DNA recombination had a great impact on the development of the discipline known as genetic engineering. This has been one of the areas with most active progress in molecular biology. Recombinant DNA methods have numerous applications. Genetic information can be introduced by DNA transfer into a cell, giving it new properties. For example, using DNA transfer, it is possible to obtain bacteria that synthesize therapeutically useful proteins or to enhance the capacity of animals or plants to produce food.

  Southern Blotting

  Even when derived from different cells or organisms, single DNA strands with complementary sequence spontaneously associate or hybridize to form a double helix when they come in contact in a medium with the appropriate composition and the right temperature. The same occurs between DNA and RNA chains when they have the sufficient base pair complementarity.

  This phenomenon of hybridization is used in several techniques to recognize specific sequences in a nucleic acid sample. A piece of DNA of known sequence (probe), complementary to that being investigated, is needed.

  Probes have different applications, such as Southern blotting (named after its inventor, Edwin Southern). In this technique, a DNA sample is cut with a restriction endonuclease and the fragments are separated by agarose gel electrophoresis, which is then immersed in an alkaline solution (NaOH) to denature the DNA. Then, DNA is transferred from the gel to another support, such as a sheet of nitrocellulose, which allows for easier manipulation. This is achieved by placing several layers of absorbent paper soaked in a concentrated salt solution inside a cuvette. The gel block containing the separated DNA is placed over the paper block and a nitrocellulose sheet is arranged at the top. Finally, several layers of dry absorbent paper are added (Fig. 21.10

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  eBook - ePub

  Molecular Biology and Genomics

  • Cornel Mulhardt(Author)
  • 2010(Publication Date)
  • Academic Press

    (Publisher)

  7

  Hybridization: How to Track Down DNA

  Publisher Summary

  Tracking down deoxyribonucleic acid (DNA) is primarily known as hybridization. In hybridization, the conditions differ considerably depending on (1) whether it is performed using a membrane with DNA or hybridizing a tissue section and (2) the stringency with which this procedure is carried out. Most frequently, Southern blot hybridizations are performed; this method is used to provide evidence for any DNA that has been transported to a particular membrane. Hybridization is usually carried out in plastic containers (with covers). The incubation is best performed in a heatable shaker with a water bath. A liquid-sparing alternative is to hybridize in plastic bags. Temperature is one of the most decisive points in hybridization. The specificity of the whole procedure can be guided by temperature. However, in principle, there is an equation to determine what temperature is correct. Another decisive point is washing correctly. It contributes to the specificity as much as the hybridization itself. The most common technique for the verification of labeled DNA is autoradiography. In nonradioactive methods of detection, fluorescein-labeled probes are used, which can be seen with ultraviolet (UV) light in a microscope or in a FluorImager. In indirect methods of detection, the probe is labeled with biotin, digoxigenin (DIG), or fluorescein and is recognized by either a specific antibody (DIG or fluorescein) or streptavidin (which binds to biotin) to which an enzyme is coupled.

   

  Tracking down DNA is primarily known as hybridization . The methods for transferring DNA and RNA to a membrane have been explained in Chapter 3 , Section 3.3

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  eBook - ePub

  Essentials of Chemical Biology

  Structure and Dynamics of Biological Macromolecules

  • Andrew D. Miller, Julian A. Tanner(Authors)
  • 2013(Publication Date)
  • Wiley

    (Publisher)

  Figure 3.14 ). If the DNA sequence is unknown, how is the probe designed? In this instance there are four general approaches.

  (a) If the amino-acid sequence is known for the desired protein, then oligodeoxynucleotide hybridisation probes may be designed with reference to the genetic code (see Chapter 1).

  (b) If the gene has previously been cloned from a related organism then a previously used probe (known as a heterologous probe) may be tried.

  (c) If a protein is known to be abundant in a particular cell/tissue, then this abundance may also be reflected in its statistical abundance within a clonal library.

  (d) If the protein of interest has been purified before, and an antibody has been raised against the protein, then the clonal library will have to be converted into an expression library (see later) and the presence of the desired protein is then screened using this antibody.

  Clearly options (a) and (b) are much preferred since they can give unambiguous identification of a gene of interest within a very small number of clones (possibly one clone) within the clonal library.

  Figure 3.14 Hybridisation in direct cloning to identify wherein the heterologous DNA of interest can be found within a clonal library. DNA from each clone is isolated by treating each colony with alkali and drying. A labelled probe is then use to hybridise to the target in order to identify in which colony the desired gene/DNA of interest can be found (Reproduced from Voe, Voet & Pratt, 1999 [Wiley] Fig. 3-31).

  3.3.2 Polymerase chain reaction

  The polymerase chain reaction (PCR) has revolutionised biological research since its invention in 1986. PCR is a method for amplifying (multiple copying) DNA, from DNA or RNA sources. PCR has many applications but an important role is the isolation and cloning of eukaryotic genes from cDNA libraries. The mechanism of PCR is as follows (Figure 3.15 ). Duplex template DNA is identified (for instance a cDNA library) and initially brought to a very high temperature (94 °C) in order that DNA strands (sense and complementary) may dissociate, a process that is often referred to as DNA melting or denaturation. The region to be amplified is then defined by the introduction of two short oligodeoxynucleotides, known as primers. When the temperature is reduced to 50–60 ° C, these primers are able to bind, or anneal, antiparallel to mutually complementary sequences in either DNA strand of the separated duplex template DNA as appropriate by Watson–Crick base pairing. The temperature is then raised to around 74 °C, optimal operating conditions for a thermostable DNA polymerase. This enzyme was originally purified from the hyperthermophile Thermus aquaticus , and hence is known as Taq polymerase.

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