Nucleic acid hybridization denaturation

For hybridization, 20 p,l of target RNA (or controls with known amounts of complementary RNA) is mixed with 10 p,I of the hybridization mix. The nucleic acid is denatured for 10 min at 80°C and then incubated, e.g., overnight at 60°C, until completion of hybridization. 

With the advent of nucleic acid hybridization as a pivotal technique in recombinant DNA technology, the theoretical and practical aspects of nucleic acid denaturation—renaturation have received renewed, more rigorous attention (11). How does a smaU piece of DNA (or RNA) find its complementary sequence from among often astronomical numbers of similar sequences Finding a 1000 base stretch in the human genome would be like finding a needle in a 2-ton haystack. 

Fig. 1. Southern blot analysis of DNA showing (a) step 1, an agarose gel containing separated restriction fragments of DNA, denoted by (—), which is immersed in NaOH to denature the double-stranded stmcture of DNA, and then transferred by capillary flow to a nitrocellulose filter. In step 2, the bound DNA is allowed to hybridize to a labeled nucleic acid probe, and the unbound probe is washed off In step 3, the filter is placed into contact with x-ray film resulting in (b) bands of exposure on the film which are detected after development and correspond to regions where the restriction fragment is...

Hybridization the process of a single-stranded nucleic acid molecule binding with a complementary single strand of nucleic acid to form a stable double-stranded molecule. Hybridization is temperature dependent, so DNA s that hybridize strongly at low temperature can be temporarily separated (denatured) by heating. 

The manufacture and processing of the protein microarray should be conducted in such a manner that the arrayed proteins remain in their native and active state. For most proteins, this usually means the hydrated state in order to avoid surface denaturation. For antibody arrays which are perhaps more forgiving than other proteins, it has been our experience that while these could be stored cold and dry, it is most important to rehydrate them prior to use. This process is in sharp contrast to the preparation of nucleic acid arrays in which strand melting or denaturahon is necessary to achieve optimal binding to the solid support. While the hybridization process is well understood and can be controlled under thermodynamic principles, the folding and renaturation of proteins on planar (microarray) surfaces is under study. 

Fig. 26. Nucleic acid structures. A The structure of the four bases in DNA, guanine (G), cytosine (C) adenine (A) and thymine (T). Uracil (U) replaces thymine (T) in RNA. B The spontaneous attraction of A for T and C for G allows the recognition of homologous sequences in aqueous solutions and the strong and specific hybridization of one sequence with its homologous sequence. C DNA forms a double helix at body temperature, which can be denatured to separate the strands by heating. D single stranded mRNA structure.

Hybridization measurements have been used in many studies of homology of nucleic acids from different species. A nucleic acid is cut (e.g., by sonic oscillation) into pieces of moderate length ( 1000 nucleotides) and is denatured. The denatured DNA fragments are mixed with denatured DNA of another species. Nucleotide sequences that are closely similar between species tend to hybridize, whereas sequences that are... 

Fig. 1. Dual nucleic acid detection. The target DNAs (A and B) are denatured and hybridized with complementary DNA probes labeled with biotin (Bio ), or digoxigenm (Dig ). Biotin and digoxigenm residues are detected, respectively, with avidin peroxidase (red) and antibody to digoxigenin labeled with alkaline phosphatase (blue).

Double-stranded DNA denatures into single strands as the temperature rises but renatures into a double-stranded structure as the temperature falls. Any two single-stranded nucleic acid molecules can form double-stranded structures (hybridize) provided that they have sufficient complementary nucleotide sequence to make the resulting hybrid stable under the reaction conditions. 

In Situ Hybridization An assay for nucleic acids on site in fixed tissue sections by the use of heat to first denature and then to reanneal with specific DNA, RNA or PNA probes. 

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