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Determination of DNA and RNA Melting Point on UV-VIS Photometer SPECORD® PLUS - Alexandra Kästner

Mar 18 2011 Comments 0

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Nucleic acids are biological molecules essential for life. Together with proteins nucleic acids make up the most important macro molecules where each is found in abundance in all living things. They allow organisms to transfer genetic information from one generation to the next. There are two types of nucleic acids: deoxyribonucleic acid, better known as DNA and ribonucleic acid, better known as RNA. Their names are derived from type of sugar, ribose, contained within the molecules. In living organisms, DNA does exist as a double helix structure. The stability is achieved by stacking and the hydrogen bonds between the bases attached to the two strands. The four bases found in DNA are adenine (abbreviated A), cytosine (C), guanine (G) and thymine (T). They form complementary pairs: The nucleotides hydrogen bond to another nucleotide base in a strand of DNA opposite to the original. This bonding is specific, and adenine always bonds to thymine (and vice versa) and guanine always bonds to cytosine (and vice versa).

Variations in pH or heating can affect to a structure modification of DNA, where the double-stranded deoxyribonucleic acid unwinds and separates into single-stranded strands through the breaking of hydrogen bonding between the bases. This denaturation process is called as DNA melting and leads to increasing of absorbance (hyperchromic effect).

From there one of the most commonly used and simplest techniques for the DNA melting point determination is spectroscopic determination by UV absorption. The absorption spectrum is recorded against the dependence of the temperature where the turning point of the graph describes the exact melting point. The temperature where at the half of the DNA exists as single strands, is called melting point (Tm).

Theory
All nucleic acids absorb strongly in the UV region due to the heterocyclic ring structure associated with each of the four bases. Typically absorption maximum is observed at a wavelength of around 260nm, although this is pH dependent. The versatility of DNA comes from this fact that the molecule is actually doublestranded. The bonding between cytosine with guanine is generally stronger than adenosine/thymine base-pairing. [1]

The amount of cytosine and guanine (called the ‘GC content’) can be estimated by measuring the temperature at which the DNA melts. Higher temperatures are associated with a high GC content. So by melting point determination it is possible to classify bacteria because the GC content in the DNA is an important fact of any organism. [2]

DNA denaturation can also be used to detect sequence differences between two nucleic acids of different origin. DNA is heated and denatured into single-stranded state, and the mixture is cooled to allow strands to rehybridise. Hybrid molecules are formed between similar sequences and any differences between those sequences will result in a disruption of the base-pairing [3].

In field of disease research rare gene mutations can be detected because mutated DNA sequences melt at lower temperatures than ‘normal’ ranges. Furthermore the process of DNA melting plays an important role in molecular biology techniques, notably in the polymerase chain reaction (PCR).


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