Compositions and methods for increasing the yields of the in...

Details Compositions and methods for increasing the yields of the in... Compositions and methods for increasing the yields of the in...

2001-09-28

2003-07-01

Weber, Jon P. (Department: 1651)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C536S025300, C536S025330

Reexamination Certificate

active

06586219

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to techniques used in molecular biology. In particular, it relates to an improved reaction mixture for use in in vitro RNA trancription and in various other enzymatic reactions in which a polynucleotide is synthesised. The reaction mixture uses high concentrations of total nucleotides, at levels that were previously thought to be inhibitory; a concentration of Mg
++
that is subsaturating with respect to the nucleotide concentration, the enzyme inorganic pyrophosphatase, and most preferrably, also Mg
++
- or Tris-nucleotides.
2. Description of the Related Art
The techniques used in the field of molecular biology have been widely and successfully applied, not only in many areas of basic research, but also in providing solutions to several medical and agricultural problems. As such, molecular biology is of great importance to scientific research, medicine, human welfare and the economy.
RNA and DNA polymerization reactions, which result in the synthesis of RNA or DNA polynucleotides, are an integral part of a variety of techniques used in molecular biology. Obviously, an increase in the yield of these reactions would be beneficial, both in saving time and expense. Such reactions include, in vitro transcription reactions, amplification techniques such as the polymerase chain reaction (PCR), self-sustained sequence replication (3SR), QB replicase and others. These reactions often employ bacteriophage RNA polymerases, such as SP6, T7 and T3, for example, in the synthesis of both radiolabeled RNA probes and unlabeled RNA.
The rate of these synthetic reactions, and the amount of product formed, is known to be limited by several factors. A common belief in the art is that these limiting factors cannot generally be overcome, and that the yield of these reactions cannot be significantly increased. For example, it is thought that nucleotide concentrations greater than 8 mM are inhibitory to in vitro transcription reactions (Gurevich et al., 1991).
In principle, the yield of reactions can generally be increased by increasing the levels of substrate(s), in this case the concentration of nucleotides used in the reaction. However, in the synthesis of polynucleotides, high levels of the nucleotide substrate are known to act as competitive inhibitors of the polymerization reaction and actually decrease the yield of the reaction. They can also lead to increases in the error rate, which is highly undesirable. The K
m
for the nucleotide substrates are quite low (ATP, 47 &mgr;M; GTP 160 &mgr;M; UTP, 60 &mgr;M; CTP 81 &mgr;M) (Chamberlin and Ring, 1973). Thus, nucleotide concentrations in the mM range are likely to be saturating, and, as mentioned above, total nucleotide concentrations above 8 mM have been reported to inhibit RNA synthesis (Gurevich et al., 1991). It is likely that this inhibition is due to competitive inhibition of the polymerase by the high level of nucleotides. This phenomenon is discussed by Chamberlin & Rhodes (1974), in regard to
E. coli
RNA polymerase.
Considering the inhibitory effects that are believed to result from using high nucleotide concentrations, the total nucleotide concentrations currently used for reportedly optimal RNA synthesis range from 1.6-16 mM (Milligan et al., 1987; Sampson & Uhlenbeck, 1988; Cunningham & Ofengand, 1990; Weitzmann, et al., 1990; Gurevich et al., 1991; Wyatt et al., 1991). It is particularly noteworthy that ‘Current Protocols in Molecular Biology’, a standard laboratory manual, recommends the lowest total nucleotide concentration, of 1.6 mM.
The substrate for polynucleotide synthetic reactions is actually a complex of the nucleotide with magnesium ions, i.e. a Mg
++
-nucleotide (Mg
++
-NTP), and therefore the magnesium concentration is also an important parameter. The conventional procedure is to routinely add magnesium at a concentration greater than the total nucleotide concentration. Indeed, the current view is that an excess of Mg
++
must be added in in vitro transcription reactions (Milligan & Uhlenbeck, 1989). It also been reported that a ratio of magnesium to total nucleotides of 1.75:1 is optimal (Wyatt et al., 1991).
The enzyme inorganic pyrophosphatase has recently been used in transcription reactions (Sampson & Uhlenbeck, 1988; Weitzmann et al., 1990; Cunningham & Ofengand, 1990), and DNA polymerase and DNA sequencing applications (Tabor & Richardson, 1990). The Cunningham and Ofengand study concluded that the pyrophosphatase increases transcription yields and also minimizes the effect of variation of magnesium concentration, although this was not believed to be due to the pyrophosphate sequestering the Mg
++
.
A further complication in polynucleotide synthesis is the fact that most phage polymerases have a low salt optima, SP6 polymerase in particular is very sensitive to even low levels of Na
+
(Butler & Chamberlin, 1982). Despite this, in most reactions of this nature nucleotide-salts are used, and in particular, Na
+
-nucleotides. Indeed, most commercially available nucleotides are the Na
+
, Li
+
, K
+
, NH
4
+
or Ba
++
salts. Increasing the levels of these nucleotides above those generally used would result in the introduction of amounts of salt which are inhibitory to the reaction.
SUMMARY OF THE INVENTION
The present invention seeks to overcome some of these and other drawbacks in the prior art by providing improved compositions for in vitro RNA trancription and various other enzymatic reactions in which a polynucleotide is synthesised. In particular, an improved reaction mixture is disclosed comprising high concentrations of total nucleotides, in the order of between about 12 mM and 40 mM, that were previously thought to be inhibitory; an effective molar concentration of Mg
++
that is subsaturating with respect to the molar total nucleotide concentration, the enzyme inorganic pyrophosphatase, and most preferrably, also Mg
++
- or Tris-nucleotides. The invention further relates to methods for employing this reaction mixture in various synthetic procedures.
As used herein, the term total nucleotide concentration is intended to refer to the total concentration of nucleotides (NTPs), i.e. the sum of the concentrations of ATP, GTP, CTP, and/or UTP, present initially in the reaction mixture when the various components of the reaction mixture have been assembled in the final volume for carrying out the reaction. Naturally, as the reaction proceeds, the nucleotides themselves will be incorporated into the polymer and so the concentration of total nucleotides will be progressively reduced from its initial value.
The process of synthesizing a polynucleotide is subject to substrate inhibition by the magnesium complexes of the various nucleotides, Mg
++
-NTPs. Higher concentrations of Mg
++
-NTPs slow the rate of polymerization as an excess of each of the other species inhibits the incorporation of the correctly matched nucleotide into the growing polymer. An important aspect of the present invention is to provide a means of increasing the total nucleotide concentration without inhibiting the reaction.
The present invention provides a reaction mixture in which the total nucleotide concentration is greater than 12 mM and not so high as to substantially inhibit the reaction to be performed. It is proposed that for most applications the upper limit nucleotide concentration will be about 100 mM, however, a higher upper limit may be appropriate under certain circumstances. For most applications, the nucleotide concentration will be between 12 mM and about 40 mM. In preferred embodiments, the total nucleotide concentration will be between about 16 mM and about 40 mM, and more preferrably, between about 20 mM and about 40 mM.
The inventors have provided other components for use in the present reaction mixture, and further adjusted the concentrations of the various constituents, such that these higher total

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