Mutation analysis by PCR and mass spectrometry

Details Mutation analysis by PCR and mass spectrometry Mutation analysis by PCR and mass spectrometry

2002-02-20

2004-06-15

Fredman, Jeffrey (Department: 1634)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S320100, C435S252800, C435S174000, C435S183000, C382S129000, C382S133000, C382S153000, C382S173000, C382S286000, C382S291000, C702S019000, C702S022000, C536S022100

Reexamination Certificate

active

06750022

ABSTRACT:

FIELD OF THE INVENTION
The invention concerns mass spectrometric analysis of known mutation sites in the genome, such as single nucleotide polymorphisms (SNPs).
BACKGROUND OF THE INVENTION
Subject of this invention is a diagnostic method for the detection of actual mutative states in the genome DNA, whereby the possible mutation site has to be known before-hand. These mutative sequence changes, compared to the standardized sequence of a “wild type”, may either be a base exchange (“point mutation”) or the introduction of nucleotides (“insertion”) or removal of nucleotides (“deletion”). Point mutations with a frequency above one percent in a population have been named “single nucleotide polymorphisms”; the abbreviation SNP has become particularly wide-spread in the recent literature. For humans, it is supposed that there are about 10 million SNPs which characterize most of the individually inherited differences between humans. They control the individual phenotypes. Roughly three million SNPs are estimated to be in the frequency range of 30 to 70 percent of the population. End of the year 2001, more than one and a quarter million SNPs were discovered and listed in the public data base NBCI of the worlwide acting SNP Consortium.
For the genome of a species, it is customary to define a “wild type” which is regarded as free of mutation, and a “mutant” which contains a mutation. Considering the frequency of mutations such as SNPs, and the equal value of mutants and wild types, the definition of the wild type is arbitrary or at least purely accidental, as already reflected in the term “polymorphism”.
Nearly all DNA mutations, including all those defined above, produce differences in the mass of the DNA segment containing the mutation in comparison to the mass of a corresponding segment of the wild type. The precise mass determination of a DNA segment can therefore be used for the determination of a mutation. Exceptions of this rule are the relatively rare “rotations”, an interchange of two bases in a sequence.
Mass spectrometry is a very powerful and precise tool for determining the mass of a bio-molecule. By using a mass spectrometric method, such as time-of-flight mass spectrometry (TOF-MS) with ionization by matrix-assisted laser desorption and ionization (MALDI), it is possible to analyze the ions for their masses. However, ionization can also be achieved using electrospray ionization (ESI), in the latter case with mass spectrometers which are frequently of a different type.
With polymerase chain reactions (PCR), using a pair of “selection primers”, i.e. single strand oligonucleotides about 20 bases long, it is possible to produce amounts in the order of billions of double-strand PCR products with a length of at least 40 base pairs in a well-known way. The production process for these oligonucleotides increases the number of products exponentially by application of temperature cycles (“thermocycles”); such processes have become known under the general term “amplification”. The mutation site can be incorporated in the products by adequately choosing the sequences of the two selection primers.
The obvious method to simply measure the mass of the PCR-amplified oligonucleotides as such by mass spectrometry, was found to be almost unworkable. The precise measurement of these DNA products with more than 40 base pairs proved itself to be almost impossible. The reasons for this are extremely low sensitivity for long DNA products because of difficult ionization, high probability of adduct formation with undefined numbers of sodium or potassium anions, and easy fragmentation of the fragile DNA products. These oligonucleotides have a poly-anionic character; each phosphate group of the DNA backbone forms an anion and has to be neutralized during ionization by a proton (which eagerly are replaced by alkali cations if present). A method therefore had to be found to provide as short oligonucleotides as possible, still containing the mutation site.
To this end, several methods of restricted, mutation-dependent primer extension using terminating derivatives of the nucleotide tri-phosphates have been developed in order to generate extended primers of approximately 12 to 25 nucleotides in length only, better suited to identify the nature of the mutation by mass spectrometry.
These methods basically consist of the following steps: Firstly, a sufficient number of copies of the DNA segment containing the mutation site is produced by PCR using a pair of selection primers. After extraction and washing, these DNA segments secondly serve as templates for the enzymatic, mutation-dependent extension of an “extension primer” by a second phase of thermocycling. In this second thermocycling phase, one to four of the nucleotide triphosphates are derivatized in such a manner that they serve as terminators for the extension, i.e., if the terminator is built in at the 3′ end, a prolongation is no longer possible because the binding site is occupied. The extension primer may be identical with one of the two selection primers; however it is regularly much better to use an extension primer which is not identical.
The extension primer is a short DNA chain of approximately 10 to 20 nucleotides and functions as a recognition sequence for the site of a possible mutation. The extension primer is synthesized with a base sequence so that it can be “hybridized” or “annealed” to the template strand, being an exact compliment to the base sequence in the vicinity of a known point mutation site. (The attachment of a complementary strand is known as “hybridization” or “annealing”).
Different types of primer extension procedures have been developed, generating either products with equal numbers of bases for mutants and wild types, differing only by the differences in weight of the different bases (9 to 40 atomic mass units as differences), or products with different numbers of bases (at least about 300 atomic mass units difference) for mutants and wild types. The latter are easier to measure by mass spectrometry, but somewhat more complicated to generate. In both cases, however, the PCR products of the first amplification cycle have to be cleaned from the nucleotide triphosphates and primers, new nucleotide triphosphates (including the terminating derivatives) and extension primers have to be added, and another set of copying thermocycles have to be applied. The final products, about 12 to 25 bases in length, again have to be thoroughly washed before mass spectrometric analysis. Primer extension procedures are complicated, using two different thermocycling and washing procedures subsequently, thus about doubling the effort of a pure PCR amplification.
The primer extension methods are widely covered by U.S. Pat. No. 6,258,538 ((H.Köster et al.).
All primer extension methods have to use rather expensive types of polymerases because not all polymerases can handle the terminating dNTP derivatives. The use of thermosequenase, especially developed for the Sanger method of sequencing, is highly recommended, more inexpensive polymerases do not correctly bind the terminators. Inexpensive polymerases, such as tac polymerase, can only be used in the first amplification by PCR.
Unfortunately, precise determination of the mass of even these relatively short primer extension oligonucleotides is still difficult. With a primer extension method delivering products with the same number of bases, the mass differences between wild type oligonucleotide and mutant oligonucleotide amount to 9 to 40 atomic mass units only. Because of the poly-anionic character of the DNA, various numbers of ubiquitous sodium (23 atomic mass units) or potassium ions (39 atomic mass units) are particularly likely to attach to the oligonucleotides (instead of protons), and so-called “adducts” are formed. The uncertainty in the degree to which the adducts are formed makes any precise mass determination exceptionally difficult-at the very least, it means that cleaning has to be extremely thorough to avoid the usually ubiquitous presence of any sodium or potassium catio

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