Research Update
Introducing Functionality
During PCR
In the fields of molecular biology and nanotechnology, enzymatic incorporation of modified dNTPs is a useful tool for the introduction of functional handles into DNA. Modified dNTP incorporation can be as simple as substituting modified dNTPs for the corresponding standard dNTPs. However, the choice of DNA polymerase, the extent of substitution and the chemical structure of the modified dNTP should be considered to achieve high density functionalization.
Structural considerations of the modified dNTP include the size of the functional group, the charge of the analog and the position of attachment to the nucleobase. In PCR, the most efficiently incorporated functionalized dNTPs contain modifications to the major groove of the nucleobase. The flexibility and length of the linker arm between the nucleobase and the functional group is also important for efficient incorporation. Some of the dNTPs TriLink offers for PCR functionalization include:
Aminoallyl-dCTP & -dUTP
Biotinylated dNTPs
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Improve Specificity with
Modified dNTPs
Many factors can affect the specificity of your PCR reaction, including nonspecific primer extension and sequence composition effects. Two types of modified dNTPs, which can be applied to improve the stringency of PCR will be discussed.
First: heat labile dNTPs, such as TriLink's CleanAmp™ dNTPs, can be employed as a universal approach to Hot Start PCR. CleanAmp™ dNTPs contain a thermolabile protecting group that keeps the reaction inactive until the initial heat cycle of PCR, where the protecting group is released to render CleanAmp™ dNTPs equivalent to their natural counterpart. Temperature-mediated control of the start of PCR reduces or eliminates off-target extensions to improve specificity.
Second: dNTPs modified to influence secondary structure formation within a PCR amplicon, have been shown to improve specificity as well. For example, secondary structure disruption is critical for successful amplification of G-C rich regions. The addition of 7-deaza-dGTP has shown significant benefit here. Other dNTPs shown to improve PCR specificity include: dITP, 2-amino-dATP, 5-propynyl-dUTP, 5-propynyl-dCTP, 5-methyl-dCTP, where dITP and 2-amino-dATP alter Watson-Crick base pairing and 5-propynyl-dUTP, 5-propynyl-dCTP and 5-methyl-dCTP increase duplex stabilty.
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Ask An Expert Featured Question
Q - I would like to link a DNA molecule to an RNA molecule. What kind of linker can
be used to link these two together? - Yun Wu, Ohio State University
A - The conjugation of two oligonucleotides together is not readily accomplished after the compounds are made. In essence, you are seeking a chemical ligation. The problem is that you would be attempting to conduct chemistry on two of the less reactive sites on the oligo – the 5′ and 3′ hydroxyls. The exocyclic amines are much more reactive. Nature activates the hydroxyls using triphoshates and then an enzyme to distort the bond angles making the reaction possible. Read More . . .
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