EP2347010A1 - QUANTITATIVE DETERMINATION OF cDNA AND GENOMIC DNA COMPRISED IN A SAMPLE - Google Patents

QUANTITATIVE DETERMINATION OF cDNA AND GENOMIC DNA COMPRISED IN A SAMPLE

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Publication number
EP2347010A1
EP2347010A1 EP09744670A EP09744670A EP2347010A1 EP 2347010 A1 EP2347010 A1 EP 2347010A1 EP 09744670 A EP09744670 A EP 09744670A EP 09744670 A EP09744670 A EP 09744670A EP 2347010 A1 EP2347010 A1 EP 2347010A1
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EP
European Patent Office
Prior art keywords
cdna
genomic dna
sequence
sequences
unique
Prior art date
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EP09744670A
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German (de)
French (fr)
Inventor
Jana Burkhardt
Johannes Boltze
Peter Ahnert
Holger Kirsten
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Fraunhofer Gesellschaft zur Foerderung der Angewandten Forschung eV
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Fraunhofer Gesellschaft zur Foerderung der Angewandten Forschung eV
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Priority to EP09744670A priority Critical patent/EP2347010A1/en
Publication of EP2347010A1 publication Critical patent/EP2347010A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to the field of molecular biology, in particular to methods in research and diagnostics with focus on the analysis of gene expression levels.
  • the present invention relates to a method of determining the amounts of cDNA and genomic DNA in a sample comprising inter alia the steps of providing a single primer pair comprised of a forward and a reverse primer, performing a PCR reaction and determining the amounts of cDNA and genomic DNA by determining the amounts of amplified unique sequences for each type of DNA.
  • the present invention also relates to a method of designing primers comprising inter alia the identification of a housekeeping gene as well as screening of the sequence of said housekeeping gene in the cDNA and the total genomic DNA for a sequence which is substantially equal regarding its composition and length but differs in at least one base.
  • the method of the invention can be used in order to determine the amounts of cDNA and genomic DNA in a sample.
  • cDNA complementary DNA
  • the total mRNA of a cell is extracted, purified and subsequently subjected to reverse transcription in order to provide cDNA.
  • Said cDNA is then analysed quantitatively wherein the amount of cDNA is proportional to the mRNA levels and thus the levels of gene expression.
  • Probes and/or primers may be designed such that they are specific for the cDNA only.
  • genomic DNA which may still be present in the sample can also hybridise or act as template. Thus, the corresponding result may be misleading since the genomic DNA still present in the sample results in signals as well.
  • genomic DNA still present in the sample may be referred to as "contaminating" DNA.
  • Genomic DNA is the only DNA present after the extraction step, and, thus, enzymes specifically degrading DNA, such as DNase I, can be used for the digestion.
  • said digestion is not necessarily 100% complete and/or the resulting sample needs to be purified again following the digestion step, which may lead to significant loss of RNA.
  • Another way of discriminating between said two DNA species is the use of sequence specific probes, which may e.g. recognize DNA regions of specific sequences present in cDNA only, such as exons spliced together (without any intronic regions which are present in genomic DNA only).
  • sequence specific probes which may e.g. recognize DNA regions of specific sequences present in cDNA only, such as exons spliced together (without any intronic regions which are present in genomic DNA only).
  • this method cannot be used for all DNA regions of interest, especially if said regions comprise long exons.
  • the use of real time PCR with appropriate primers and optionally with taqMan probes has also been described for discriminating between cDNA and genomic DNA.
  • the ratio of cDNA to genomic DNA in the final sample used for cDNA analysis is determined (after the step of reverse transcription). With said ratio known, the expression levels determined can be fitted to the levels originating from the cDNA only and thus corrected.
  • Said method comprises inter alia the provision of a single primer pair and the amplification of unique sequences in the cDNA and the genomic DNA by a PCR reaction.
  • the method of the present invention can thus be used to determine the amounts of cDNA and genomic DNA in a sample.
  • a method of determining the amounts of cDNA and genomic DNA in a sample comprises at least the steps of:
  • step a) comprising cDNA and genomic DNA of the method above is provided outside the human or animal body. This also applies to all samples comprising DNA mentioned throughout the description, if it is not explicitly mentioned.
  • Said sample may comprise genomic DNA in trace amounts only or no genomic DNA at all; however, the result that no genomic DNA can be determined in the sample represents a valuable result as well since it indicates that no further type of DNA except cDNA is present in the sample.
  • said forward primer hybridizes to the 5' end of the unique sequences and said reverse primer hybridizes to the 3' end of the unique sequences such that said primers hybridise to complementary strands of the DNA and are directed in 5' to 3' direction towards each other.
  • said two primers hybridise completely to the sequences in the cDNA and the genomic DNA flanking said unique sequences.
  • said two primers may also only partially hybridise to said sequences.
  • a primer may hybridise completely to the sequence in one type of DNA but only partially to the sequence in the other type of DNA.
  • said two unique sequences are of substantially equal length ranging from about 50 bp to about 1000 bp with about 150 bp being preferred.
  • said two unique sequences are of substantially equal composition ranging in a GC content in % GC from about 30% to about 70%.
  • the reaction kinetics of the amplification reaction of the two unique sequences are substantially equal.
  • the conditions of the amplification reaction are optimised with regard to the substantially equal length and substantially equal composition of the two unique sequences. For the latter point, this can e.g. refer to the elongation cycle of the PCR reaction, wherein preferably 2 min/kb per elongation cycle are set up when regular Taq polymerase is used.
  • the amounts of both amplified sequences are determined by methods which allow distinguishing between said two amplified sequences by their difference in at least one base. Furthermore, said methods allow quantifying said amplified sequences. Said methods can be chosen from methods such as sequencing methods, PCR methods such as real time PCR, TaqMan methods, mass spectrometry, Temperature Gradient Gel Electrophoresis (TGGE), Denaturating Gradient Gel Electrophoresis (DGGE) and genotyping methods, e.g. mass spectrometry based genotyping.
  • said methods are coupled, such as the use of single base extension (SBE)-PCR in order to provide different products depending on one of the at least one differing nucleotides followed by mass spectrometry to determine the amounts of the different SBE-PCR products and optionally comprise a purification step.
  • SBE single base extension
  • the amplified sequences are purified prior to the determination of their amounts via methods selected from the group of methods comprising gel filtration, DNA precipitation and PCR product purification.
  • the amplified sequences are purified prior to the determination of their amounts by gel filtration using an agarose gel followed by band excision and gel extraction.
  • said unique sequence in the cDNA as well as said unique sequence in the genomic DNA are each present in equal copies in either the cDNA or the genomic DNA.
  • said unique sequence in the cDNA is present only once in the cDNA and said unique sequence in the genomic DNA is present only once in the genomic DNA.
  • said unique sequence in the cDNA and said unique sequence in the genomic DNA are present in different copies in either the cDNA or the genomic DNA. This may e.g. result from several duplication steps of the corresponding DNA sequence. In this embodiment, it is important that the correction factor for these different copies is taken into account when determining the amounts of the two unique sequences in step e) of the method of the present invention.
  • the copy number of the unique sequences is, in both cases, i.e. for the cDNA and the genomic DNA, determined by performing PCR reactions using purified DNA as template, in each case over a concentration range of the corresponding DNA with fixed primer concentrations.
  • a titration analysis of either purified cDNA or of purified genomic DNA is done in order to determine the order of the reaction and thus the copy number(s) in each the template.
  • a method of designing primers comprises at least the steps of:
  • said coding sequence in the cDNA and the total genomic DNA are screened for a sequence, which is substantially equal regarding its composition and length but differs in at least one base and also differs substantially regarding its composition and length from the genomic sequence of the gene identified in step a).
  • said primers are in a particularly preferred embodiment of the invention in 5' to 3' direction a forward primer of the sequence GAGCACAGAGCCTCGCCT (SEQ ID No. 2) and a reverse primer of the sequence TCGTCGCCCACATAGGAA (SEQ ID No. 3). Said primer pair may be used in accordance with the first object of the present invention.
  • the method of the present invention with the preferred embodiments as set out above can be used in order to determine the amounts of cDNA and genomic DNA in a sample.
  • Figure 1 cDNA sequence of ⁇ -actin. The sequence is the result of a reverse transcription of the ⁇ -actin mRNA. Sequence in bold italic: unique sequence in the cDNA of ⁇ -actin chosen for amplification; underlined sequences: sequences at the 5' and 3' ends of the unique sequence corresponding to forward and reverse priming sites.
  • Figure 2 Alignment of the primer sequences to genomic DNA sequences.
  • A alignment of two possible frames for the forward primer using the ⁇ -actin cDNA sequence and genomic sequences on chromosomes as depicted.
  • B alignment of a possible frame for the reverse primer using the ⁇ -actin cDNA sequence and genomic sequences on chromosomes as depicted.
  • Small letters mismatches; note that sequences on cDNA and chromosome 5 are identical.
  • A Unique sequence in the ⁇ -actin cDNA (also depicted in figure 1).
  • B Unique sequence on chromosome 5. 5' and 3' ends corresponding to the priming sites are underlined and italic. Bases differing between sequence A and B are given in small letters. Note that 18 bases differ.
  • Bold priming sites for an SBE-primer used for identification of either sequence A or B (reverse complement sequence of the SBE-primer).
  • FIG. 5 MALDI-TOF analysis of SBE-PCR products.
  • the three spectra of the samples after an SBE-PCR are depicted (the sequence of the SBE-primer is depicted in figure 3). Only one product can be detected in the reaction comprising genomic DNA (SBE + T), whereas in the cDNA a product of an additional mass corresponding to the cDNA product can be detected as well (SBE + C). Expected masses of the corresponding products are depicted on the right.
  • cDNA samples A-G: cDNA preparations derived from different individual human EBV transfected B-cell lines. In each spectrum, the unextended
  • the peak intensities correspond to the amounts of PCR product in the sample and are proportional to the relative amounts of cDNA and genomic DNA, respectively (depicted here in %).
  • the inventors have found that it is possible to determine the amounts of cDNA and genomic DNA in a sample using a method comprising inter alia the provision of a primer pair flanking unique sequences in the cDNA and in the genomic DNA.
  • the inventors have found a method of designing an appropriate primer pair comprising inter alia the screening of a sequence in the cDNA and the genomic DNA for a substantially equal sequence.
  • the method of the present invention is cost-effective, fast and requires in certain embodiments standard laboratory equipment only. Furthermore, it provides a reliable result as to whether and to what a extent a cDNA sample comprises genomic DNA.
  • the terms “about” and “approximately” denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question.
  • the term typically indicates a deviation from the indicated numerical value of +/- 10 % and preferably +1- 5 %.
  • the present invention relates to a method of determining the amounts of cDNA and genomic DNA in a sample.
  • Said method comprises at least the steps of: a) providing a sample comprising cDNA and genomic DNA as templates; b) providing a forward and a reverse primer wherein said two primers hybridise to sequences flanking a unique sequence in the cDNA and wherein said two primers hybridize to sequences flanking a unique sequence in the genomic DNA wherein said two unique sequences are substantially equal regarding their composition and length but differ in at least one base; c) performing a PCR reaction using the templates of step a) and the primers of step b); d) determining the amount of amplified sequence derived from the unique sequence in the cDNA and the amount of amplified sequence derived from the unique sequence in the genomic DNA; e) assigning the amounts of cDNA and genomic DNA in said sample wherein the amount of sequence derived from the cDNA represents the amount of cDNA, and the amount of sequence derived from
  • Said method may be used to determine the amounts of cDNA and genomic DNA in a sample.
  • General terms as used in the description of the present invention will now be explained and defined in the following section of the description.
  • the term “amount” as used herein is preferably used in the meaning of “relative amount” vs. "absolute amount”.
  • the term “absolute amount” refers to the amount or molecular weight, respectively, of DNA in a sample expressed in g or preferably in ⁇ g. With the volume of the sample in 1 or ⁇ l and the absolute amount known, the concentration of the DNA in a sample can be calculated and expressed, e.g. in ⁇ g / ⁇ l.
  • the term “relative amount” refers to the amount of one type of DNA in relation (and thus relative) to the total amount of DNA in the sample, which may be expressed as 100%. Thus, if only one type of DNA is present in a sample, the relative amount of said DNA in the sample is 100%. If two types of DNA are present in a ratio of 1 : 10, the relative amount of the first species will be 10% with the second at 90%.
  • the relative amounts can be deduced from the intensities of signals and/or signal to noise ratios, such as applied to peaks in MALDI-TOF or peaks in sequencing reactions. If more than one signal for a type of DNA is detected, the sum of intensities of the more than one signal corresponds to the total relative amount of DNA.
  • the amounts of DNA determined in step e) thus represent in preferred embodiments relative amounts of the corresponding DNA present in the sample. With these relative amounts at hand, one can easily determine a correction factor which may be necessary in order to correct for signals derived from genomic DNA.
  • RNA as used herein describes a synthetic, so-called “complementary” (with respect to the RNA template) DNA, which typically has been produced via an enzymatic reaction using reverse transcriptase.
  • an RNA preparation is typically the initial template.
  • RNA is extracted from cells which are subject to analysis according to standard protocols known to the skilled person.
  • Said RNA preparation is provided outside the human or animal body, whereas the cells analysed may originate from a human or animal body.
  • the present invention is not used for any treatment steps or in vivo applications.
  • Said initial RNA template is then transcribed by the reverse transcriptase in a RNA/DNA double-strand, which is in the following step then processed into a DNA/DNA double-strand.
  • any organism or cell may be subject to an RNA extraction step.
  • cells of any tissue or of any differentiation state may also be subject to investigation.
  • the method of the present invention can be applied to any cDNA preparation originating from RNA preparations of any origin.
  • RNA in the cell may be used. However, it can be preferred to purify and isolate mRNA (m for messenger RNA) in order to analyze e.g. expression profiles of genes coding for proteins. However, it should be noted that also other products of gene expression are known, such as small interfering RNA molecules or the like. Depending on the stability and the purification protocols, it is also possible to purify and enrich said other RNA types and analyse their expression levels via reverse transcription into cDNA followed by a quantification of the cDNA.
  • mRNA messenger RNA
  • other products of gene expression such as small interfering RNA molecules or the like.
  • cDNA libraries which originate from collections of cell types, tissues, organisms and the like.
  • cDNA libraries may e.g. derive from normal tissue and from cancerous tissue.
  • up- or down- regulated genes may be identified and appropriate therapy may be initiated.
  • the amount of cDNA in the sample is proportional to the amount of RNA since the process of reverse transcription is proportional to the starting material.
  • genomic DNA refers to initial DNA present in a cell, which is subject to analysis. "Genomic DNA” as used herein does not refer to synthetic DNA such as cDNA but to naturally occurring DNA. As outlined above, cells are provided outside the human or animal body and RNA is extracted from the cells. After the process of cell disruption and/or RNA extraction, genomic DNA may still be present in the preparation of RNA used for the cDNA preparation.
  • cDNA The analysis of cDNA can be employed in a wide range of applications.
  • the step following the cDNA preparation is the quantitative determination of cDNA.
  • any genomic DNA may, however, lead to wrong results, since the method used may also amplify regions in the genomic DNA which are similar to sequences in the cDNA. This will, in the end, lead to an artificial expression level.
  • the genomic DNA may only account for traces of DNA in the total sample. This, however, can still influence the results of experiments analysing the cDNA, e.g. if the expression level is low and only very little amounts of RNA are present. Therefore, it is essential to know the amounts of said two types of DNA in the sample.
  • template or "DNA template” is used in the context of a PCR reaction and is known to the skilled person.
  • DNA template comprising two complementary DNA strands, i.e. one leading and one lagging strand, needs to be present.
  • a "forward primer” represents a primer used for amplification processes during a PCR reaction.
  • the forward primer is always designed in complementary fashion to the reverse primer such that the amplification directions are opposite and the corresponding two reaction products can anneal in a next step during the PCR reaction.
  • the forward primer is e.g. complementary to the lagging strand. However, it may also be complementary to the leading strand if the reverse primer is designed accordingly.
  • a forward primer is usually designed at the 5' end of the sequence to be amplified and thus flanks the 5 'end of the PCR product.
  • a "reverse primer” is comprised of nucleotides on the complementary strand of the forward primer.
  • the reverse primer will flank the PCR product at the 3' end and, thus, it is designed such that its 5 'end starts with the last base of the sequence to be amplified with the direction in 3' towards the 5' end of the sequence to be amplified.
  • the sequence amplified using the reverse primer will thus be complementary to the sequence of the reaction using the forward primer.
  • PCR reaction will be explained in the following in a simplified and schematic view.
  • At least one DNA template and a forward as well a reverse primer as set out above are provided in a reaction tube.
  • a DNA polymerase, dNTP's and corresponding buffer conditions are provided in said tube.
  • double stranded DNA is melting and one leading and one lagging strand are therefore present in the sample.
  • the temperature is lowered to an annealing temperature and held for several seconds up to about 1 minute, the two primers anneal to the corresponding strands of the DNA template, i.e.
  • Typical annealing temperatures range from about 40 to about 65 0 C.
  • the elongation step the temperature is increased to the optimal working temperature for the polymerase used (about 65 to about 75 0 C), such that the polymerase elongates the primers using the complementary strand as template. Depending on the polymerase, certain incubation times are chosen for this step, e.g. 2 minutes per 1 kb to be amplified.
  • the temperature is again increased to a denaturing temperature such that the double stranded products of the first elongation reaction are separated.
  • PCR reactions comprise about 20 to about 40 cycles. In the context of the present invention, it is preferred that a standard PCR reaction is carried out.
  • hybridising or “annealing” as used herein refers to base pairing between two DNA strands by hydrogen-bonding between an A and a T as well as a G and C, respectively, of the two strands. If one single stranded DNA sequences is comprised of exactly the reverse complement sequence as a second one, said two single stranded DNA molecules will hybridise with each other and form double-stranded DNA.
  • said short primer will hybridise to the complementary sequence in the genomic DNA.
  • This process can also be referred to as the annealing of the primer. It is important to note that hybridisation does not necessarily mean that both strands are completely complementary. For the present invention it is, however, preferred that the forward and the reverse primer anneal completely the sequences in the cDNA and in the genomic DNA. One mismatch or a low number of mismatches may, however, be tolerated as long as specific annealing takes place.
  • the conditions of the PCR reaction can be optimised to still allow for specific annealing. Particularly the temperature chosen for the annealing process is important in this regard. Also, the GC-content of the primers plays an important role in this process since G and C form three hydrogen bonds with each other whereas A and T form only two hydrogen bonds.
  • the term "unique sequence” refers to a sequence of a defined length and composition, which is preferably present only once either in the cDNA or in the genomic DNA. "Length” refers to the numbers of bases, whereas “composition” refers to the sequence itself, i.e. the order of nucleotides and the number of each single nucleotide in the total sequence. Typically, the number of each single nucleotide is expressed as GC-content or % GC in the total sequence. In certain embodiments, the GC-content may also be referred to as the composition.
  • a unique sequence may comprise 10 nucleotides and have the following order of nucleotides GCCGCATTT A, resulting in a GC-content of 50%.
  • the term "unique” as used with respect to sequence does, however, not exclude that a second copy of said unique sequence may also be present in the corresponding DNA. This will be explained in detail below.
  • flanking means that a specific sequence, preferably of 20 bases, which is located at the 5' end and the 3' end of a unique sequence. It needs to be understood that a sequence which is flanking a unique sequence can also be comprised in the unique sequence. However, in case said flanking sequences are comprised in the unique sequences of either the cDNA or the genomic DNA, the at least one differing base will preferably not be located in said flanking sequences. Thus, the flanking sequences are preferably identical in the cDNA and the genomic DNA.
  • substantially equal as used herein is meant to describe a state, which is almost identical to a second state but can be discriminated from the first state.
  • two unique sequences may have e.g. exactly the same length, but may differ with respect to their composition, i.e. with regard to their base order and/or composition. Said difference may manifest in one base only, which, however, is sufficient for the present invention. Insertions of several bases in one sequence only resulting in different lengths of the two unique sequences can also be detected by the method of the present invention. In this case, composition and length would be different.
  • the length of the sequences may preferably be in a range from about 50 bases to about 1000 bases.
  • the sequences can be about 500 bases long, preferably about 250 bases, more preferably about 200 bases and most preferably about 150 bases. However, it needs to be understood that the length of the two sequences does not need to be equal, but only substantially equal as explained in the following.
  • substantially equal thus describes in certain embodiments two sequences differing in their lengths by about 50%, about 30%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2% or about 1%.
  • substantially equal describes two sequences differing in their lengths by about 30% at most, preferably by about 20% at most, more preferably by about 15% at most, and most preferably by about 10% at most.
  • substantially equal describes two sequences differing in their lengths by about 5% at most.
  • the two unique sequences may also differ in their GC-content by about 75% to about 1%, with about 10% to about 1% being especially preferred.
  • the two unique sequences located either in the cDNA or in the genomic DNA are substantially equal but differ in at least one base.
  • the shorter the unique sequences the less differing bases should be present.
  • the conditions of the PCR reaction are preferably adapted to the unique sequences to be amplified. Thus, if long unique sequences are selected, the elongation cycle will preferably be longer than when a short sequence is amplified only. Furthermore, the person skilled in the art is aware that other factors such as the polymerase or factors such as the salt concentration also influence the PCR reaction. However, standard PCR reactions are preferred.
  • the PCR reaction may lead to a variety of products. Since the primers are used in excess, the templates are, however, limiting. Thus, if the two unique sequences are known, the identification and quantification is directed and adapted to the amplified sequences originating from said two sequences, even with a background of other products. As long as said amplified products can be identified and quantified, further PCR products of the PCR reaction can be neglected.
  • Methods in order to determine the amounts of PCR products can be selected from the group of methods comprising sequencing methods, PCR methods such as real time PCR, TaqMan methods, mass spectrometry, Temperature Gradient Gel Electrophoresis (TGGE), Denaturating Gradient Gel Electrophoresis (DGGE) and genotyping methods such as mass spectrometry based genotyping.
  • Said methods can optionally be coupled as described in the example section below wherein an SBE- PCR is performed followed by identification of the products by mass spectrometry.
  • any method known to the skilled person which is able to discriminate between the two unique sequences and which is able to quantify the products can be used.
  • PCR products Prior to the analysis of the two amplified unique sequences differing in at least one base, it may be necessary and preferred to purify the PCR products. Any purification method known to the skilled person, such as desalting methods or precipitation techniques of PCR products can be used. Size exclusion (e.g. gel filtration) may also be an appropriate method since the size of the resulting PCR products to be analysed is known from the beginning and, thus, a corresponding method may be used and optimised in order to purify and enrich corresponding products. In this regards, chromatography methods such as agarose gels or columns may be used as well as affinity chromatography methods comprising DNA binding membranes or beads and the like. It is especially preferred to purify the amplified sequences by gel filtration using an agarose gel followed by band excision and gel extraction.
  • Any purification method known to the skilled person such as desalting methods or precipitation techniques of PCR products can be used.
  • Size exclusion e.g. gel filtration
  • chromatography methods such as agarose
  • purification methods may also be used which are e.g. included in the protocol and workflow of the analysis method chosen (such as in the protocol of a genotyping method).
  • Said purification step comprised in such a protocol may be subsequent to the analyzing step itself.
  • the two unique sequences are preferably present in equal copies, most preferably only once, in the cDNA and in the genomic DNA. It is therefore ensured that the two sequences differing in at least one base are amplified in an equal ratio and the corresponding amounts of cDNA and genomic DNA can directly be assigned according to the amounts of amplified sequences.
  • the unique sequence in the cDNA is most preferably present only once in the cDNA, whereas the unique sequence differing in at least one base in the genomic DNA may be present in two or more copies.
  • the amplification reaction using two identical templates in the genomic DNA will result in a higher number of amplified sequences. Therefore, said higher number needs to be corrected in order to be comparable to the amplified sequences originating from the cDNA.
  • This may be achieved by performing PCR calibration reactions wherein either purified genomic DNA of the cell type to be analyzed or purified cDNA of said cell type is incubated as template over a concentration range with fixed concentrations of the primer pair (typically in excess).
  • concentrations of the primer pair typically in excess.
  • the unique sequences chosen are blasted against the whole genomic DNA sequence of the cell to be analyzed in order to identify identical copies.
  • the inventors have also found a method of designing primers which may be used according to the present invention.
  • a housekeeping gene i.e. a gene which is stably expressed from the genomic DNA of the cell subject to analysis. This may be done using software and databases available at NCBI providing information on the expression pattern of different organisms and/or tissues and/or cells as well as large numbers of publications on this matter.
  • Preferred housekeeping genes are e.g. genes implicated in the structure of a cell such as actins, tubulins, or genes implicated in the metabolism of a cell such as GAPDH, or genes implicated in other essential cell mechanisms such as rRNAs, and so on.
  • the corresponding mRNA will be present in the cell, which means that the cDNA will also be present in the cDNA preparation.
  • the overall goal of the experiment is the quantitative analysis of gene expression wherein said analysis comprises a step of analysing the expression level of a housekeeping gene for normalisation reasons, it is especially preferred that said exact same housekeeping gene is also used for the method of the present invention, i.e. the cDNA sequence of said housekeeping gene is chosen for screening as outlined in the next paragraph.
  • the cDNA sequence of said housekeeping gene is screened against the total genomic DNA for a common shorter sequence which is substantially equal regarding its composition and length but differs in at least one base. Screening may be done by manual comparison of the cDNA and silenced duplicated sequences of the housekeeping gene in the genomic DNA and/or by using software such as human Blat, Blast 2 sequences or Office programs (e.g.Word, Excel).
  • the cDNA sequence chosen cannot be identical to the sequence of the corresponding gene on the genomic DNA. Furthermore, said sequence of the gene in the genomic DNA and said cDNA sequences should not be substantially equal and thus should be different, e.g. by introns which are present in the gene on the genomic DNA only.
  • said two unique sequences are thus substantially equal regarding their composition and length but differ in at least one base and also differ substantially from the genomic sequence of the stably expressed gene chosen, i.e. the housekeeping gene. Said aspect can be important in order to avoid cross-amplification.
  • the corresponding forward and reverse primers are designed at the 5 ' and 3 ' ends of said sequences.
  • the primers not necessarily need to correspond to the ends of the unique sequences. Said ends (and thus the primers) may also be shifted towards each other resulting in shorter sequences to be amplified for the benefit of selecting an optimal primer pair. Of course, only one end may be shifted as well.
  • primers are most suited which display almost identical melting temperatures in the range of between 57°C and 63°C (and thus a GC content of between 30% and 70%).
  • any secondary structures present in a primer may be disadvantageous and result in the selection of a new primer.
  • Software such as Netprimer or Calcdalton is typically used by the skilled person to design and analyse appropriate primers.
  • the overall purpose of the experiments as set out below was to determine the amount of genomic DNA present in several cDNA preparations of different human EBV transfected B-cell lines.
  • the cDNA preparations were obtained using a standard protocol for this purpose, namely the PeqGOLD TriFastTM protocol (Peqlab) for RNA isolation and the RevertAidTM H Minus M-MuLV Reverse Transcriptase Kit (Fermentas) for reverse transcription.
  • Example 1 Identification of two substantially equal unique sequences (one in the cDNA and one in the genomic DNA) and primer design
  • the goal was to identify a sequence in the cDNA, which is also present in the genomic DNA, but differs in at least one base from the cDNA sequence.
  • the analysis was done using standard software available at NCBI such as human Blat (http://genome.ucsc.edu/cgi-bin/hgBlat), Blast 2 Sequences
  • ⁇ -actin As housekeeping gene expressed in B-cells and thus present in the cDNA, ⁇ -actin was chosen.
  • the cDNA sequence (here also referred to as CDS) originating from the corresponding mRNA sequence of ⁇ -actin as a result of reverse transcription is given in Figure 1 (SEQ ID No. 1). It comprises 1794 bases.
  • the corresponding gene on the genomic DNA must not be identical to the CDS chosen.
  • the ⁇ -actin gene located on chromosome 7 (SEQ ID No. 31) was compared to the CDS. Due to introns present in the gene, said gene comprises 3436 bases and is thus much longer than the CDS.
  • the CDS of ⁇ -actin was then compared to sequences present in the total genomic DNA.
  • the cDNA and the genomic DNA were screened for similar sequences. Such sequences were identified in the following chromosomes: 5, 18, 15, 9, 5, X, 10, 3 and 19.
  • the sequence chosen is shown in the CDS of ⁇ -actin in italic bold in figure 1.
  • sequences of 20 bases at the 5' and 3' ends of the identified sequence with approximately identical melting temperatures were chosen.
  • Figure 2B shows the possible frame for the reverse primer. Again, only the complementary reverse primer sequence for the CDS and chromosome 5 are identical and flank the identified sequence at the 3' side. Mismatches are given in small letters in Figure 2.
  • DNA-RNA for (SEQ ID No. 2): GAGCACAGAGCCTCGCCT (58.19 0 C)
  • DNA-RNA rev (SEQ ID No. 3): TCGTCGCCCACATAGGAA (57.55 0 C)
  • Said primers (for the reverse primer the corresponding reverse complementary sequence) are underlined in figures 1 and 3 and flank a sequence of the ⁇ -actin CDS as given in Figure 3A (SEQ ID No. 28) and a sequence on chromosome 5 of the genomic DNA as given in Figure 3B (SEQ ID No. 29). Said sequences differ in 18 nucleotides, indicated by small letters.
  • Both sequences have an identical length of 229 bp.
  • the CDS sequence shows a content of 63,83 % GC, whereas the sequence on chromosome 5 shows a content of 69,36 % GC.
  • both sequences have an substantially equal content of GC resulting in very similar behaviour during an amplification reaction regarding their annealing and melting reactions.
  • the two primers of course also anneal to sequences in the gene coding for ⁇ -actin on chromosome 7.
  • the sequence flanked in this region is comprised of 1223 bp.
  • said 1223 bp fragment is not substantially equal regarding the composition and length and can easily be excluded and discriminated from the other two 229 bp sequences, e.g. by adapting the PCR reaction to not amplify larger products and/or by appropriate purification methods as set out below.
  • Example 2 amplification and purification of the unique sequences, exemplary described for one of the cDNA samples
  • the following protocol was optimised with respect to the detection of the unique sequences by SBE-PCR. However, it may be adapted to any other detection method such as DNA sequencing.
  • the templates used were cDNAs of the different human EBV trans fected B-cell lines (A to G), the corresponding genomic DNA only (B), and TE -buffer (C).
  • the PCR reaction was set up as follows:
  • the mastermix was split in 15 tubes, 0.3 ⁇ l of each primer and 1 ⁇ l template were added according to the following scheme:
  • Tubes 1-7 template A to G cDNA
  • Tubes 8-14 template A to G gDNA
  • Tube 15 control without template (TE buffer instead)
  • the PCR reaction was carried out using the following parameters:
  • Figure 4 shows the samples of tubes 1, 8 and 15 corresponding to lines 9, 10 and 11 as indicated.
  • the sample using TE buffer as control did not result in any product.
  • the products of the expected size were detected (see square).
  • Example 3 identification of unique sequences and determination of their amounts exemplary described for one of the cDNA samples
  • the reverse complementary sequence of the SBE primer is indicated in both sequences shown in figure 3A and 3B in bold; thus, the primer is directed with its 3 ' end towards a differing base (an A in the genomic DNA and a C in the cDNA). It was designed according to the guidelines for primer design in SBE-reactions with the software PrimExtend. To additionally check for secondary structures and melting temperatures the software Netprimer was used. As 18 bases differ between the two unique sequences, other possible SBE-primer locations lie at other differing bases and could, in principle, also be used.
  • the primer had the following sequence in 5' to 3 'direction (wherein L indicates a light inducible cleavage site):
  • the primer will be extended with a C if cDNA is present and with a T if genomic DNA is present.
  • the PCR reaction was set up as follows:
  • the SBE reaction was carried out using the following parameters:
  • Figure 5 shows the results for tube 1, 8 and 15:
  • the sample with TE buffer as template did not lead to any peaks corresponding to the expected masses of SBE- products (lowest graph).
  • the sample comprising genomic B-cell DNA only resulted in a peak corresponding to a mass expected for a T as the extended base, whereas the cDNA preparation resulted in the exact same peak (indicating that genomic DNA is still present in the sample) in addition to a peak corresponding to a product with a C as the extended base (indicating cDNA).
  • the cDNA peak in the sample had an intensity of 3 arbitrary units compared to 1 unit for the genomic DNA peak. Thus, the total amount of DNA present in the sample corresponds to 4 units. 3/4 or 75% of said total DNA is cDNA with 25% genomic DNA still present in the sample.
  • Example 4 determination of the amount of genomic DNA present in other cDNA samples of different human B-cell lines
  • the samples correspond to:
  • B cDNA preparation from human an EBV transformed B-cell line M21
  • C cDNA preparation from human an EBV transformed B-cell line M23
  • D cDNA preparation from human an EBV transformed B-cell line M33
  • E cDNA preparation from human an EBV transformed B-cell line M35
  • F cDNA preparation from human an EBV transformed B-cell line M40
  • G cDNA preparation from human an EBV transformed B-cell line M45

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Abstract

The present invention relates to a method of determining the amounts of cDNA and genomic DNA in a sample using a PCR reaction, the use thereof and to a method of designing primers accordingly.

Description

QUANTITATIVE DETERMINATION OF cDNA AND GENOMIC DNA
COMPRISED IN A SAMPLE
FIELD OF THE INVENTION
The present invention relates to the field of molecular biology, in particular to methods in research and diagnostics with focus on the analysis of gene expression levels.
With respect to such methods, the present invention relates to a method of determining the amounts of cDNA and genomic DNA in a sample comprising inter alia the steps of providing a single primer pair comprised of a forward and a reverse primer, performing a PCR reaction and determining the amounts of cDNA and genomic DNA by determining the amounts of amplified unique sequences for each type of DNA.
For the method of the present invention, it is crucial to design said primer pair accordingly.
Thus, the present invention also relates to a method of designing primers comprising inter alia the identification of a housekeeping gene as well as screening of the sequence of said housekeeping gene in the cDNA and the total genomic DNA for a sequence which is substantially equal regarding its composition and length but differs in at least one base.
The method of the invention can be used in order to determine the amounts of cDNA and genomic DNA in a sample. BACKGROUND OF THE INVENTION
The analysis of cDNA (complementary DNA) samples originating from mRNA of various organism, tissues or cells of various differentiation states represents a major analysis method in order to determine inter alia the expression levels of genes.
In case gene expression levels are analysed, the total mRNA of a cell is extracted, purified and subsequently subjected to reverse transcription in order to provide cDNA. Said cDNA is then analysed quantitatively wherein the amount of cDNA is proportional to the mRNA levels and thus the levels of gene expression.
Most of the methods used today for the quantification of cDNA rely on hybridization and/or amplification reactions, such as the use of specific probes as hybridising targets in arrays or the use of specific primers in real time PCR methods. Probes and/or primers may be designed such that they are specific for the cDNA only.
However, not all of said methods are specific for cDNA only and, thus, genomic DNA which may still be present in the sample can also hybridise or act as template. Thus, the corresponding result may be misleading since the genomic DNA still present in the sample results in signals as well. In this context, genomic DNA still present in the sample may be referred to as "contaminating" DNA.
Methods in order to get rid of such contaminating genomic DNA have been established.
First of all, it is possible to specifically digest DNA after the RNA extraction step but before the step of reverse transcription.
Genomic DNA is the only DNA present after the extraction step, and, thus, enzymes specifically degrading DNA, such as DNase I, can be used for the digestion. However, said digestion is not necessarily 100% complete and/or the resulting sample needs to be purified again following the digestion step, which may lead to significant loss of RNA. Another way of discriminating between said two DNA species is the use of sequence specific probes, which may e.g. recognize DNA regions of specific sequences present in cDNA only, such as exons spliced together (without any intronic regions which are present in genomic DNA only). However, this method cannot be used for all DNA regions of interest, especially if said regions comprise long exons. Furthermore, the use of real time PCR with appropriate primers and optionally with taqMan probes has also been described for discriminating between cDNA and genomic DNA. Using said methods, the ratio of cDNA to genomic DNA in the final sample used for cDNA analysis is determined (after the step of reverse transcription). With said ratio known, the expression levels determined can be fitted to the levels originating from the cDNA only and thus corrected.
However, methods used today for said correction are time-consuming and cost- intensive by relying on elaborate machines such as real time PCR machines or devices such as DNA-arrays. In this regard, it should be mentioned that real time PCR machines and corresponding taqMan probes as well as all equipment necessary for DNA arrays do not represent standard techniques available in every laboratory. Furthermore, two different sets of primers, one being specific for the DNA and the other being specific for the RNA, are typically used resulting in different efficiencies of the two different reactions. Thus, there is the need for a quick, simple and cost-effective method in order to determine the amount of cDNA and the amount of genomic DNA present in a sample wherein the amount of genomic DNA may be regarded as "contamination" depending on the further analysis methods chosen for characterising the amount of cDNA and/or the cDNA itself. OBJECTS AND SUMMARY OF THE INVENTION
It is an objective of the present invention to provide a method for determining the amount of cDNA and the amount of genomic DNA in a sample. Said method comprises inter alia the provision of a single primer pair and the amplification of unique sequences in the cDNA and the genomic DNA by a PCR reaction.
Furthermore, it is an objective of the present invention to provide a method of designing primers which can be used according to the method described above.
The method of the present invention can thus be used to determine the amounts of cDNA and genomic DNA in a sample.
According to one aspect of the invention, a method of determining the amounts of cDNA and genomic DNA in a sample is provided. Said method comprises at least the steps of:
a) providing a sample comprising cDNA and genomic DNA as templates; b) providing a forward and a reverse primer wherein said two primers hybridise to sequences flanking a unique sequence in the cDNA and wherein said two primers hybridize to sequences flanking a unique sequence in the genomic DNA wherein said two unique sequences are substantially equal regarding their composition and length but differ in at least one base; c) performing a PCR reaction using the templates of step a) and the primers of step b); d) determining the amount of amplified sequence derived from the unique sequence in the cDNA and the amount of amplified sequence derived from the unique sequence in the genomic DNA; e) assigning the amounts of cDNA and genomic DNA in said sample wherein the amount of sequence derived from the cDNA represents the amount of cDNA, and the amount of sequence derived from the genomic DNA represents the amount of genomic DNA, and the two amounts represent the amount of total DNA.
The sample of step a) comprising cDNA and genomic DNA of the method above is provided outside the human or animal body. This also applies to all samples comprising DNA mentioned throughout the description, if it is not explicitly mentioned.
Said sample may comprise genomic DNA in trace amounts only or no genomic DNA at all; however, the result that no genomic DNA can be determined in the sample represents a valuable result as well since it indicates that no further type of DNA except cDNA is present in the sample.
In a preferred embodiment of the aspect mentioned above, said forward primer hybridizes to the 5' end of the unique sequences and said reverse primer hybridizes to the 3' end of the unique sequences such that said primers hybridise to complementary strands of the DNA and are directed in 5' to 3' direction towards each other.
In an also preferred embodiment of the present invention, said two primers hybridise completely to the sequences in the cDNA and the genomic DNA flanking said unique sequences. However, in other embodiments, said two primers may also only partially hybridise to said sequences. In one embodiment, a primer may hybridise completely to the sequence in one type of DNA but only partially to the sequence in the other type of DNA.
In a further preferred embodiment of the method of the present invention, said two unique sequences are of substantially equal length ranging from about 50 bp to about 1000 bp with about 150 bp being preferred.
In another preferred embodiment of the method of the present invention, said two unique sequences are of substantially equal composition ranging in a GC content in % GC from about 30% to about 70%.
In also preferred embodiment of the present invention, the reaction kinetics of the amplification reaction of the two unique sequences are substantially equal. Furthermore, in a preferred embodiment, the conditions of the amplification reaction are optimised with regard to the substantially equal length and substantially equal composition of the two unique sequences. For the latter point, this can e.g. refer to the elongation cycle of the PCR reaction, wherein preferably 2 min/kb per elongation cycle are set up when regular Taq polymerase is used.
In a further preferred embodiment of the present invention, the amounts of both amplified sequences are determined by methods which allow distinguishing between said two amplified sequences by their difference in at least one base. Furthermore, said methods allow quantifying said amplified sequences. Said methods can be chosen from methods such as sequencing methods, PCR methods such as real time PCR, TaqMan methods, mass spectrometry, Temperature Gradient Gel Electrophoresis (TGGE), Denaturating Gradient Gel Electrophoresis (DGGE) and genotyping methods, e.g. mass spectrometry based genotyping. In preferred embodiments, said methods are coupled, such as the use of single base extension (SBE)-PCR in order to provide different products depending on one of the at least one differing nucleotides followed by mass spectrometry to determine the amounts of the different SBE-PCR products and optionally comprise a purification step. In another preferred embodiment of the present invention, the amplified sequences are purified prior to the determination of their amounts via methods selected from the group of methods comprising gel filtration, DNA precipitation and PCR product purification.
In a particularly preferred embodiment of the invention, the amplified sequences are purified prior to the determination of their amounts by gel filtration using an agarose gel followed by band excision and gel extraction.
In a further preferred embodiment of the method of the present invention, said unique sequence in the cDNA as well as said unique sequence in the genomic DNA are each present in equal copies in either the cDNA or the genomic DNA.
In an especially preferred embodiment, said unique sequence in the cDNA is present only once in the cDNA and said unique sequence in the genomic DNA is present only once in the genomic DNA.
However, in another embodiment of the method of the present invention, said unique sequence in the cDNA and said unique sequence in the genomic DNA are present in different copies in either the cDNA or the genomic DNA. This may e.g. result from several duplication steps of the corresponding DNA sequence. In this embodiment, it is important that the correction factor for these different copies is taken into account when determining the amounts of the two unique sequences in step e) of the method of the present invention.
In an also preferred embodiment, the copy number of the unique sequences is, in both cases, i.e. for the cDNA and the genomic DNA, determined by performing PCR reactions using purified DNA as template, in each case over a concentration range of the corresponding DNA with fixed primer concentrations. Thus, in this embodiment, a titration analysis of either purified cDNA or of purified genomic DNA is done in order to determine the order of the reaction and thus the copy number(s) in each the template.
In a further object of the present invention, which is linked to the first object as mentioned above, a method of designing primers is provided, wherein said primers are used according to the method as set out above. The method of designing primers comprises at least the steps of:
a) Identifying a gene which is stably expressed from the genomic DNA the cDNA originates from, such that the coding sequence of said gene is present in the cDNA; b) Screening said coding sequence in the cDNA and the total genomic DNA for a sequence which is substantially equal regarding its composition and length but differs in at least one base; c) Designing a forward primer covering about 20 bases in 5' to 3' direction at the 5' end of said sequence which is substantially equal; d) Designing a reverse primer covering about 20 bases in 5 ' to 3 ' direction on the complementary strand at the 3' end of said sequence which is substantially equal.
In a preferred embodiment regarding the further object of the invention, said coding sequence in the cDNA and the total genomic DNA are screened for a sequence, which is substantially equal regarding its composition and length but differs in at least one base and also differs substantially regarding its composition and length from the genomic sequence of the gene identified in step a).
With respect to said further object of the invention, said primers are in a particularly preferred embodiment of the invention in 5' to 3' direction a forward primer of the sequence GAGCACAGAGCCTCGCCT (SEQ ID No. 2) and a reverse primer of the sequence TCGTCGCCCACATAGGAA (SEQ ID No. 3). Said primer pair may be used in accordance with the first object of the present invention.
Overall, the method of the present invention with the preferred embodiments as set out above can be used in order to determine the amounts of cDNA and genomic DNA in a sample.
DESCRIPTION OF THE FIGURES
If not explicitly mentioned, all DNA sequences of the figures are given in 5' to 3' direction.
Figure 1: cDNA sequence of β-actin. The sequence is the result of a reverse transcription of the β-actin mRNA. Sequence in bold italic: unique sequence in the cDNA of β-actin chosen for amplification; underlined sequences: sequences at the 5' and 3' ends of the unique sequence corresponding to forward and reverse priming sites.
Figure 2: Alignment of the primer sequences to genomic DNA sequences. A: alignment of two possible frames for the forward primer using the β-actin cDNA sequence and genomic sequences on chromosomes as depicted. B: alignment of a possible frame for the reverse primer using the β-actin cDNA sequence and genomic sequences on chromosomes as depicted. Small letters: mismatches; note that sequences on cDNA and chromosome 5 are identical.
Figure 3:
Unique sequences amplified in the cDNA and on chromosome 5. A: Unique sequence in the β-actin cDNA (also depicted in figure 1). B: Unique sequence on chromosome 5. 5' and 3' ends corresponding to the priming sites are underlined and italic. Bases differing between sequence A and B are given in small letters. Note that 18 bases differ. Bold: priming sites for an SBE-primer used for identification of either sequence A or B (reverse complement sequence of the SBE-primer).
Figure 4:
Agarose gel of PCR products. As described in the example section, three different samples were used as targets in the PCR reactions: a cDNA preparation of a human B-cell line (PCR products in lane 9); genomic DNA of human B-cell line (PCR products lane 10); TE -buffer as control (PCR product in lane 11). Note that the expected products have a size of 229 bp.
Figure 5: MALDI-TOF analysis of SBE-PCR products. The three spectra of the samples after an SBE-PCR are depicted (the sequence of the SBE-primer is depicted in figure 3). Only one product can be detected in the reaction comprising genomic DNA (SBE + T), whereas in the cDNA a product of an additional mass corresponding to the cDNA product can be detected as well (SBE + C). Expected masses of the corresponding products are depicted on the right.
Figure 6:
MALDI-TOF analysis of SBE-PCR products of cDNA preparations of different human cell lines. cDNA samples: A-G: cDNA preparations derived from different individual human EBV transfected B-cell lines. In each spectrum, the unextended
SBE-primer, the "cDNA" and the "gDNA" (g for genomic) peak are highlighted.
The peak intensities (in arbitrary units) correspond to the amounts of PCR product in the sample and are proportional to the relative amounts of cDNA and genomic DNA, respectively (depicted here in %). DETAILED DESCRIPTION OF THE INVENTION
The inventors have found that it is possible to determine the amounts of cDNA and genomic DNA in a sample using a method comprising inter alia the provision of a primer pair flanking unique sequences in the cDNA and in the genomic DNA.
Furthermore, the inventors have found a method of designing an appropriate primer pair comprising inter alia the screening of a sequence in the cDNA and the genomic DNA for a substantially equal sequence.
The method of the present invention is cost-effective, fast and requires in certain embodiments standard laboratory equipment only. Furthermore, it provides a reliable result as to whether and to what a extent a cDNA sample comprises genomic DNA.
While describing in detail exemplary embodiments of the present invention, definitions important for understanding the present invention are provided.
As used in the specification and the appended claims, the singular form of "a" and "an" also includes the respective plurals, unless the context clearly dictates otherwise.
In the context of the present invention, the terms "about" and "approximately" denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of +/- 10 % and preferably +1- 5 %.
It needs to be understood that the term "comprising" is not limiting. For the purpose of the present invention, the term "consisting of is considered to be a preferred embodiment of the term "comprising of ". If, hereinafter, a group is defined as comprising at least a certain number of embodiments, this is also meant to encompass a group that preferably consists of these embodiments only.
As has been set out above, the present invention relates to a method of determining the amounts of cDNA and genomic DNA in a sample. Said method comprises at least the steps of: a) providing a sample comprising cDNA and genomic DNA as templates; b) providing a forward and a reverse primer wherein said two primers hybridise to sequences flanking a unique sequence in the cDNA and wherein said two primers hybridize to sequences flanking a unique sequence in the genomic DNA wherein said two unique sequences are substantially equal regarding their composition and length but differ in at least one base; c) performing a PCR reaction using the templates of step a) and the primers of step b); d) determining the amount of amplified sequence derived from the unique sequence in the cDNA and the amount of amplified sequence derived from the unique sequence in the genomic DNA; e) assigning the amounts of cDNA and genomic DNA in said sample wherein the amount of sequence derived from the cDNA represents the amount of cDNA, and the amount of sequence derived from the genomic DNA represents the amount of genomic DNA, and the two amounts represent the amount of total DNA.
Said method may be used to determine the amounts of cDNA and genomic DNA in a sample. General terms as used in the description of the present invention will now be explained and defined in the following section of the description.
The term "amount" as used herein is preferably used in the meaning of "relative amount" vs. "absolute amount". The term "absolute amount" refers to the amount or molecular weight, respectively, of DNA in a sample expressed in g or preferably in μg. With the volume of the sample in 1 or μl and the absolute amount known, the concentration of the DNA in a sample can be calculated and expressed, e.g. in μg / μl. The term "relative amount" refers to the amount of one type of DNA in relation (and thus relative) to the total amount of DNA in the sample, which may be expressed as 100%. Thus, if only one type of DNA is present in a sample, the relative amount of said DNA in the sample is 100%. If two types of DNA are present in a ratio of 1 : 10, the relative amount of the first species will be 10% with the second at 90%.
However, by determining the relative amounts of e.g. two types of DNA present in a sample with the overall absolute amount of DNA in the sample known (determined e.g. by standard concentration assays such as UV-determination at 260 nm known to the skilled person), it is easily possible to determine the absolute amounts of each type of DNA in the sample. The determination of the absolute amounts may, however, not be necessary since the relative amounts are sufficient for correcting e.g. expression levels.
Depending on the methods used in the present invention, the relative amounts can be deduced from the intensities of signals and/or signal to noise ratios, such as applied to peaks in MALDI-TOF or peaks in sequencing reactions. If more than one signal for a type of DNA is detected, the sum of intensities of the more than one signal corresponds to the total relative amount of DNA. The amounts of DNA determined in step e) thus represent in preferred embodiments relative amounts of the corresponding DNA present in the sample. With these relative amounts at hand, one can easily determine a correction factor which may be necessary in order to correct for signals derived from genomic DNA.
The term "cDNA" as used herein describes a synthetic, so-called "complementary" (with respect to the RNA template) DNA, which typically has been produced via an enzymatic reaction using reverse transcriptase. For this purpose, an RNA preparation is typically the initial template. Thus, in a first step, RNA is extracted from cells which are subject to analysis according to standard protocols known to the skilled person. Said RNA preparation is provided outside the human or animal body, whereas the cells analysed may originate from a human or animal body. However, the present invention is not used for any treatment steps or in vivo applications. Said initial RNA template is then transcribed by the reverse transcriptase in a RNA/DNA double-strand, which is in the following step then processed into a DNA/DNA double-strand.
In principle, any organism or cell may be subject to an RNA extraction step. However, for the present invention, it may be preferred to extract RNA from cells selected from the group of cells comprising human cells, animal cells, bacterial cells and fungal cells. If an organism comprised of differentiated cells is chosen for analysis, cells of any tissue or of any differentiation state may also be subject to investigation. Thus, there is no restriction regarding cell types for the method of the present invention. The method of the present invention can be applied to any cDNA preparation originating from RNA preparations of any origin.
Any RNA in the cell may be used. However, it can be preferred to purify and isolate mRNA (m for messenger RNA) in order to analyze e.g. expression profiles of genes coding for proteins. However, it should be noted that also other products of gene expression are known, such as small interfering RNA molecules or the like. Depending on the stability and the purification protocols, it is also possible to purify and enrich said other RNA types and analyse their expression levels via reverse transcription into cDNA followed by a quantification of the cDNA.
There are also cDNA libraries known, which originate from collections of cell types, tissues, organisms and the like. Thus, cDNA libraries may e.g. derive from normal tissue and from cancerous tissue. Using a quantitative analysis, up- or down- regulated genes may be identified and appropriate therapy may be initiated.
The amount of cDNA in the sample is proportional to the amount of RNA since the process of reverse transcription is proportional to the starting material.
The term "genomic DNA" as used herein refers to initial DNA present in a cell, which is subject to analysis. "Genomic DNA" as used herein does not refer to synthetic DNA such as cDNA but to naturally occurring DNA. As outlined above, cells are provided outside the human or animal body and RNA is extracted from the cells. After the process of cell disruption and/or RNA extraction, genomic DNA may still be present in the preparation of RNA used for the cDNA preparation.
The analysis of cDNA can be employed in a wide range of applications. For the analysis of gene expression, the step following the cDNA preparation is the quantitative determination of cDNA. In this process, which may be done e.g. using real time PCR methods or DNA arrays, any genomic DNA may, however, lead to wrong results, since the method used may also amplify regions in the genomic DNA which are similar to sequences in the cDNA. This will, in the end, lead to an artificial expression level. The genomic DNA may only account for traces of DNA in the total sample. This, however, can still influence the results of experiments analysing the cDNA, e.g. if the expression level is low and only very little amounts of RNA are present. Therefore, it is essential to know the amounts of said two types of DNA in the sample.
The term "template" or "DNA template" is used in the context of a PCR reaction and is known to the skilled person. For a regular PCR reaction, a DNA template comprising two complementary DNA strands, i.e. one leading and one lagging strand, needs to be present.
A "forward primer" represents a primer used for amplification processes during a PCR reaction. The forward primer is always designed in complementary fashion to the reverse primer such that the amplification directions are opposite and the corresponding two reaction products can anneal in a next step during the PCR reaction. In the example outlined below, the forward primer is e.g. complementary to the lagging strand. However, it may also be complementary to the leading strand if the reverse primer is designed accordingly. A forward primer is usually designed at the 5' end of the sequence to be amplified and thus flanks the 5 'end of the PCR product.
Correspondingly, a "reverse primer" is comprised of nucleotides on the complementary strand of the forward primer. The reverse primer will flank the PCR product at the 3' end and, thus, it is designed such that its 5 'end starts with the last base of the sequence to be amplified with the direction in 3' towards the 5' end of the sequence to be amplified. The sequence amplified using the reverse primer will thus be complementary to the sequence of the reaction using the forward primer.
The term "PCR reaction" will be explained in the following in a simplified and schematic view. At least one DNA template and a forward as well a reverse primer as set out above are provided in a reaction tube. Additionally, a DNA polymerase, dNTP's and corresponding buffer conditions (such as salt concentrations and the like) are provided in said tube. In the initial denaturing step at a temperature of about 95°C of up to several minutes, double stranded DNA is melting and one leading and one lagging strand are therefore present in the sample. During the next step wherein the temperature is lowered to an annealing temperature and held for several seconds up to about 1 minute, the two primers anneal to the corresponding strands of the DNA template, i.e. one to the leading and the other primer to the lagging strand. Typical annealing temperatures range from about 40 to about 65 0C. In the next, step, the elongation step, the temperature is increased to the optimal working temperature for the polymerase used (about 65 to about 75 0C), such that the polymerase elongates the primers using the complementary strand as template. Depending on the polymerase, certain incubation times are chosen for this step, e.g. 2 minutes per 1 kb to be amplified. Finally, in the last step, the temperature is again increased to a denaturing temperature such that the double stranded products of the first elongation reaction are separated. Following this denaturation step, the next cycle comprising annealing, elongation and denaturing is then initiated. Typically, PCR reactions comprise about 20 to about 40 cycles. In the context of the present invention, it is preferred that a standard PCR reaction is carried out.
The term "hybridising" or "annealing" as used herein refers to base pairing between two DNA strands by hydrogen-bonding between an A and a T as well as a G and C, respectively, of the two strands. If one single stranded DNA sequences is comprised of exactly the reverse complement sequence as a second one, said two single stranded DNA molecules will hybridise with each other and form double-stranded DNA.
In case one of said molecules is a short primer only and the second molecule is a single stranded long DNA sequence (such as a denaturated genomic DNA), said short primer will hybridise to the complementary sequence in the genomic DNA. This process can also be referred to as the annealing of the primer. It is important to note that hybridisation does not necessarily mean that both strands are completely complementary. For the present invention it is, however, preferred that the forward and the reverse primer anneal completely the sequences in the cDNA and in the genomic DNA. One mismatch or a low number of mismatches may, however, be tolerated as long as specific annealing takes place. If a mismatch is present due to the sequence chosen, the conditions of the PCR reaction can be optimised to still allow for specific annealing. Particularly the temperature chosen for the annealing process is important in this regard. Also, the GC-content of the primers plays an important role in this process since G and C form three hydrogen bonds with each other whereas A and T form only two hydrogen bonds.
As used herein, the term "unique sequence" refers to a sequence of a defined length and composition, which is preferably present only once either in the cDNA or in the genomic DNA. "Length" refers to the numbers of bases, whereas "composition" refers to the sequence itself, i.e. the order of nucleotides and the number of each single nucleotide in the total sequence. Typically, the number of each single nucleotide is expressed as GC-content or % GC in the total sequence. In certain embodiments, the GC-content may also be referred to as the composition. Thus, a unique sequence may comprise 10 nucleotides and have the following order of nucleotides GCCGCATTT A, resulting in a GC-content of 50%. The term "unique" as used with respect to sequence does, however, not exclude that a second copy of said unique sequence may also be present in the corresponding DNA. This will be explained in detail below.
As used herein, the term "flanking" means that a specific sequence, preferably of 20 bases, which is located at the 5' end and the 3' end of a unique sequence. It needs to be understood that a sequence which is flanking a unique sequence can also be comprised in the unique sequence. However, in case said flanking sequences are comprised in the unique sequences of either the cDNA or the genomic DNA, the at least one differing base will preferably not be located in said flanking sequences. Thus, the flanking sequences are preferably identical in the cDNA and the genomic DNA.
The term "substantially equal" as used herein is meant to describe a state, which is almost identical to a second state but can be discriminated from the first state. This means that two unique sequences may have e.g. exactly the same length, but may differ with respect to their composition, i.e. with regard to their base order and/or composition. Said difference may manifest in one base only, which, however, is sufficient for the present invention. Insertions of several bases in one sequence only resulting in different lengths of the two unique sequences can also be detected by the method of the present invention. In this case, composition and length would be different.
The length of the sequences may preferably be in a range from about 50 bases to about 1000 bases. The sequences can be about 500 bases long, preferably about 250 bases, more preferably about 200 bases and most preferably about 150 bases. However, it needs to be understood that the length of the two sequences does not need to be equal, but only substantially equal as explained in the following.
"Substantially equal" thus describes in certain embodiments two sequences differing in their lengths by about 50%, about 30%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2% or about 1%.
In preferred embodiments of the present invention, "substantially equal" describes two sequences differing in their lengths by about 30% at most, preferably by about 20% at most, more preferably by about 15% at most, and most preferably by about 10% at most. In an especially preferred embodiment of the present invention, "substantially equal" describes two sequences differing in their lengths by about 5% at most. With respect to their composition, the two unique sequences may also differ in their GC-content by about 75% to about 1%, with about 10% to about 1% being especially preferred.
The term "substantially" with regard to the PCR reaction carried out for the method of the present invention can also describe a situation, in which the reaction kinetics of the amplification reactions are more or less equal. This will also depend on the conditions used during the PCR reaction.
As already set out above, the two unique sequences located either in the cDNA or in the genomic DNA, are substantially equal but differ in at least one base. In general, it is noteworthy that the shorter the unique sequences, the less differing bases should be present. The longer the two unique sequences, the more bases can differ since this will overall not substantially affect their similarity regarding composition and length as well as the subsequent PCR amplification.
The conditions of the PCR reaction are preferably adapted to the unique sequences to be amplified. Thus, if long unique sequences are selected, the elongation cycle will preferably be longer than when a short sequence is amplified only. Furthermore, the person skilled in the art is aware that other factors such as the polymerase or factors such as the salt concentration also influence the PCR reaction. However, standard PCR reactions are preferred.
For the present invention, it needs to be understood that the PCR reaction may lead to a variety of products. Since the primers are used in excess, the templates are, however, limiting. Thus, if the two unique sequences are known, the identification and quantification is directed and adapted to the amplified sequences originating from said two sequences, even with a background of other products. As long as said amplified products can be identified and quantified, further PCR products of the PCR reaction can be neglected. Methods in order to determine the amounts of PCR products can be selected from the group of methods comprising sequencing methods, PCR methods such as real time PCR, TaqMan methods, mass spectrometry, Temperature Gradient Gel Electrophoresis (TGGE), Denaturating Gradient Gel Electrophoresis (DGGE) and genotyping methods such as mass spectrometry based genotyping. Said methods can optionally be coupled as described in the example section below wherein an SBE- PCR is performed followed by identification of the products by mass spectrometry. In general, any method known to the skilled person which is able to discriminate between the two unique sequences and which is able to quantify the products can be used.
Prior to the analysis of the two amplified unique sequences differing in at least one base, it may be necessary and preferred to purify the PCR products. Any purification method known to the skilled person, such as desalting methods or precipitation techniques of PCR products can be used. Size exclusion (e.g. gel filtration) may also be an appropriate method since the size of the resulting PCR products to be analysed is known from the beginning and, thus, a corresponding method may be used and optimised in order to purify and enrich corresponding products. In this regards, chromatography methods such as agarose gels or columns may be used as well as affinity chromatography methods comprising DNA binding membranes or beads and the like. It is especially preferred to purify the amplified sequences by gel filtration using an agarose gel followed by band excision and gel extraction.
However, the skilled person is aware that purification methods may also be used which are e.g. included in the protocol and workflow of the analysis method chosen (such as in the protocol of a genotyping method). Thus, when following the experimental protocol chosen for analysis, it might not be necessary to carry out an additional purification since said step is part of the protocol. Said purification step comprised in such a protocol may be subsequent to the analyzing step itself. As already outlined above, the two unique sequences are preferably present in equal copies, most preferably only once, in the cDNA and in the genomic DNA. It is therefore ensured that the two sequences differing in at least one base are amplified in an equal ratio and the corresponding amounts of cDNA and genomic DNA can directly be assigned according to the amounts of amplified sequences.
However, said two sequences may also be present in different copies. The unique sequence in the cDNA is most preferably present only once in the cDNA, whereas the unique sequence differing in at least one base in the genomic DNA may be present in two or more copies. Obviously, the amplification reaction using two identical templates in the genomic DNA will result in a higher number of amplified sequences. Therefore, said higher number needs to be corrected in order to be comparable to the amplified sequences originating from the cDNA. This may be achieved by performing PCR calibration reactions wherein either purified genomic DNA of the cell type to be analyzed or purified cDNA of said cell type is incubated as template over a concentration range with fixed concentrations of the primer pair (typically in excess). The skilled person is aware of such calibration reactions in order to determine the order of the reaction and thus an optional correction factor. Preferably, the unique sequences chosen are blasted against the whole genomic DNA sequence of the cell to be analyzed in order to identify identical copies.
The inventors have also found a method of designing primers which may be used according to the present invention.
In a first step, a housekeeping gene, i.e. a gene which is stably expressed from the genomic DNA of the cell subject to analysis, is identified. This may be done using software and databases available at NCBI providing information on the expression pattern of different organisms and/or tissues and/or cells as well as large numbers of publications on this matter. Preferred housekeeping genes are e.g. genes implicated in the structure of a cell such as actins, tubulins, or genes implicated in the metabolism of a cell such as GAPDH, or genes implicated in other essential cell mechanisms such as rRNAs, and so on. Thus, the corresponding mRNA will be present in the cell, which means that the cDNA will also be present in the cDNA preparation.
If the overall goal of the experiment is the quantitative analysis of gene expression wherein said analysis comprises a step of analysing the expression level of a housekeeping gene for normalisation reasons, it is especially preferred that said exact same housekeeping gene is also used for the method of the present invention, i.e. the cDNA sequence of said housekeeping gene is chosen for screening as outlined in the next paragraph.
In a second step, the cDNA sequence of said housekeeping gene is screened against the total genomic DNA for a common shorter sequence which is substantially equal regarding its composition and length but differs in at least one base. Screening may be done by manual comparison of the cDNA and silenced duplicated sequences of the housekeeping gene in the genomic DNA and/or by using software such as human Blat, Blast 2 sequences or Office programs (e.g.Word, Excel).
In this regard, it should be noted that the cDNA sequence chosen cannot be identical to the sequence of the corresponding gene on the genomic DNA. Furthermore, said sequence of the gene in the genomic DNA and said cDNA sequences should not be substantially equal and thus should be different, e.g. by introns which are present in the gene on the genomic DNA only.
In a preferred embodiment of the invention, said two unique sequences are thus substantially equal regarding their composition and length but differ in at least one base and also differ substantially from the genomic sequence of the stably expressed gene chosen, i.e. the housekeeping gene. Said aspect can be important in order to avoid cross-amplification.
Finally, after having identified two unique sequences differing in at least one base, the corresponding forward and reverse primers are designed at the 5 ' and 3 ' ends of said sequences. In general, the primers not necessarily need to correspond to the ends of the unique sequences. Said ends (and thus the primers) may also be shifted towards each other resulting in shorter sequences to be amplified for the benefit of selecting an optimal primer pair. Of course, only one end may be shifted as well. The skilled person is aware that primers are most suited which display almost identical melting temperatures in the range of between 57°C and 63°C (and thus a GC content of between 30% and 70%). Furthermore, the skilled person is aware that any secondary structures present in a primer may be disadvantageous and result in the selection of a new primer. Software such as Netprimer or Calcdalton is typically used by the skilled person to design and analyse appropriate primers.
EXAMPLES
The overall purpose of the experiments as set out below was to determine the amount of genomic DNA present in several cDNA preparations of different human EBV transfected B-cell lines. The cDNA preparations were obtained using a standard protocol for this purpose, namely the PeqGOLD TriFast™ protocol (Peqlab) for RNA isolation and the RevertAid™ H Minus M-MuLV Reverse Transcriptase Kit (Fermentas) for reverse transcription. Example 1 : Identification of two substantially equal unique sequences (one in the cDNA and one in the genomic DNA) and primer design
The goal was to identify a sequence in the cDNA, which is also present in the genomic DNA, but differs in at least one base from the cDNA sequence. The analysis was done using standard software available at NCBI such as human Blat (http://genome.ucsc.edu/cgi-bin/hgBlat), Blast 2 Sequences
(http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi) and Office related software (Word, Excel), as well as the mRNA sequences available via Entrez Gene
(http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene) and the FASTA program (http://www.ebi.ac.uk/Tools/fasta/index.html) as well as the sequence of the human genome project at NCBI.
As housekeeping gene expressed in B-cells and thus present in the cDNA, β-actin was chosen. The cDNA sequence (here also referred to as CDS) originating from the corresponding mRNA sequence of β-actin as a result of reverse transcription is given in Figure 1 (SEQ ID No. 1). It comprises 1794 bases.
Obviously, the corresponding gene on the genomic DNA must not be identical to the CDS chosen. Thus, in a first step, the β-actin gene located on chromosome 7 (SEQ ID No. 31) was compared to the CDS. Due to introns present in the gene, said gene comprises 3436 bases and is thus much longer than the CDS.
The CDS of β-actin was then compared to sequences present in the total genomic DNA. The cDNA and the genomic DNA were screened for similar sequences. Such sequences were identified in the following chromosomes: 5, 18, 15, 9, 5, X, 10, 3 and 19. The sequence chosen is shown in the CDS of β-actin in italic bold in figure 1. In the next step, sequences of 20 bases at the 5' and 3' ends of the identified sequence with approximately identical melting temperatures were chosen.
Figure 2A shows two possibilities for annealing of the forward primer to the identified sequences on the different chromosomes, "frame 1" and "frame 2". However, only the forward primer sequences for the CDS (BACT = β-actin) and chromosome 5 are identical and flank the identified sequence at the 5' side. Figure 2B shows the possible frame for the reverse primer. Again, only the complementary reverse primer sequence for the CDS and chromosome 5 are identical and flank the identified sequence at the 3' side. Mismatches are given in small letters in Figure 2.
Thus, the following primers with given melting temperatures were chosen:
DNA-RNA for (SEQ ID No. 2): GAGCACAGAGCCTCGCCT (58.19 0C) DNA-RNA rev (SEQ ID No. 3): TCGTCGCCCACATAGGAA (57.55 0C)
Said primers (for the reverse primer the corresponding reverse complementary sequence) are underlined in figures 1 and 3 and flank a sequence of the β-actin CDS as given in Figure 3A (SEQ ID No. 28) and a sequence on chromosome 5 of the genomic DNA as given in Figure 3B (SEQ ID No. 29). Said sequences differ in 18 nucleotides, indicated by small letters.
Both sequences have an identical length of 229 bp. The CDS sequence shows a content of 63,83 % GC, whereas the sequence on chromosome 5 shows a content of 69,36 % GC. Thus, both sequences have an substantially equal content of GC resulting in very similar behaviour during an amplification reaction regarding their annealing and melting reactions. The two primers of course also anneal to sequences in the gene coding for β-actin on chromosome 7. However, the sequence flanked in this region is comprised of 1223 bp. Clearly, said 1223 bp fragment is not substantially equal regarding the composition and length and can easily be excluded and discriminated from the other two 229 bp sequences, e.g. by adapting the PCR reaction to not amplify larger products and/or by appropriate purification methods as set out below.
Example 2: amplification and purification of the unique sequences, exemplary described for one of the cDNA samples
The following protocol was optimised with respect to the detection of the unique sequences by SBE-PCR. However, it may be adapted to any other detection method such as DNA sequencing.
The templates used were cDNAs of the different human EBV trans fected B-cell lines (A to G), the corresponding genomic DNA only (B), and TE -buffer (C).
The PCR reaction was set up as follows:
The mastermix was split in 15 tubes, 0.3 μl of each primer and 1 μl template were added according to the following scheme:
Tubes 1-7: template A to G cDNA
Tubes 8-14 : template A to G gDNA
Tube 15: control without template (TE buffer instead)
The PCR reaction was carried out using the following parameters:
After the PCR reaction, the samples were subjected to an agarose gel DNA electrophoresis to control for expected products and their sizes. Figure 4 shows the samples of tubes 1, 8 and 15 corresponding to lines 9, 10 and 11 as indicated. As expected, the sample using TE buffer as control did not result in any product. However, using either the cDNA or the genomic DNA sample, the products of the expected size were detected (see square).
Example 3: identification of unique sequences and determination of their amounts exemplary described for one of the cDNA samples
In order to identify the unique sequences, a standard SBE-PCR reaction according to the GenoSNIP method was performed. The reverse complementary sequence of the SBE primer is indicated in both sequences shown in figure 3A and 3B in bold; thus, the primer is directed with its 3 ' end towards a differing base (an A in the genomic DNA and a C in the cDNA). It was designed according to the guidelines for primer design in SBE-reactions with the software PrimExtend. To additionally check for secondary structures and melting temperatures the software Netprimer was used. As 18 bases differ between the two unique sequences, other possible SBE-primer locations lie at other differing bases and could, in principle, also be used. The primer had the following sequence in 5' to 3 'direction (wherein L indicates a light inducible cleavage site):
biotin - ATCATCATC(L)ATGGTGAGCTGG
Correspondingly, the primer will be extended with a C if cDNA is present and with a T if genomic DNA is present.
The PCR reaction was set up as follows:
6 μl mastermix were added to tubes 1-15 as set out above. Digested PCR product was used as template in a separate reaction performed as control for the SBE- reaction.
The SBE reaction was carried out using the following parameters:
Denaturation "b0:04:00 W C Nx
Denaturation 00:00:10 94° C
Primerhybridisation 00:00:30 60° C 44x
Elongation 00:00:10 72° C
Purification of the products was done according to the GenoSNP manual
Finally, the products of the SBE-PCR reaction were analysed using MALDI-TOF for their molecular masses. Figure 5 shows the results for tube 1, 8 and 15: The sample with TE buffer as template did not lead to any peaks corresponding to the expected masses of SBE- products (lowest graph). The sample comprising genomic B-cell DNA only resulted in a peak corresponding to a mass expected for a T as the extended base, whereas the cDNA preparation resulted in the exact same peak (indicating that genomic DNA is still present in the sample) in addition to a peak corresponding to a product with a C as the extended base (indicating cDNA).
Quantification was done using the peak intensities. The cDNA peak in the sample had an intensity of 3 arbitrary units compared to 1 unit for the genomic DNA peak. Thus, the total amount of DNA present in the sample corresponds to 4 units. 3/4 or 75% of said total DNA is cDNA with 25% genomic DNA still present in the sample.
Example 4: determination of the amount of genomic DNA present in other cDNA samples of different human B-cell lines
The samples correspond to:
A: cDNA preparation from human an EBV transformed B-cell line M 19
B: cDNA preparation from human an EBV transformed B-cell line M21 C: cDNA preparation from human an EBV transformed B-cell line M23 D: cDNA preparation from human an EBV transformed B-cell line M33 E: cDNA preparation from human an EBV transformed B-cell line M35 F: cDNA preparation from human an EBV transformed B-cell line M40 G: cDNA preparation from human an EBV transformed B-cell line M45
The method was carried out as described above and results for the amounts of cDNA and genomic DNA (gDNA), respectively, were obtained as indicated in figure 6.

Claims

1. Method of determining the amounts of cDNA and genomic DNA in a sample comprising at least the steps of: a) providing a sample comprising cDNA and genomic DNA as templates; b) providing a forward and a reverse primer wherein said two primers hybridise to sequences flanking a unique sequence in the cDNA and wherein said two primers hybridize to sequences flanking a unique sequence in the genomic DNA wherein said two unique sequences are substantially equal regarding their composition and length but differ in at least one base; c) performing a PCR reaction using the templates of step a) and the primers of step b); d) determining the amount of amplified sequence derived from the unique sequence in the cDNA and the amount of amplified sequence derived from the unique sequence in the genomic DNA; e) assigning the amounts of cDNA and genomic DNA in said sample wherein the amount of sequence derived from the cDNA represents the amount of cDNA, and the amount of sequence derived from the genomic DNA represents the amount of genomic DNA, and the two amounts represent the amount of total DNA.
2. Method according to claim 1 wherein said forward primer hybridises to the 5' end of the unique sequences and said reverse primer hybridises to the 3' end of the unique sequences such that said primers hybridize to complementary strands of the DNA and are directed in 5' to 3' direction towards each other.
3. Method according to claims 1 and 2 wherein said two primers hybridise completely to the sequences in the cDNA and the genomic DNA flanking said unique sequences.
4. Method according to any of the preceding claims wherein said two unique sequences are of substantially equal length ranging from about 50 bp to about 1000 bp with about 150 bp being preferred.
5. Method according to any of the preceding claims wherein said two unique sequences are of substantially equal composition ranging in a GC content in %
GC from about 30% to about 70%.
6. Method according to any of the preceding claims wherein the reaction kinetics of the amplification reaction of the two unique sequences are substantially equal and the conditions of the amplification reaction are optimised with regard to the substantially equal length and substantially equal composition of the two unique sequences.
7. Method according to any of the preceding claims wherein the amounts of both amplified sequences are determined by methods which allow to distinguish between said two amplified sequences by their difference in at least one base and which allow to quantify said amplified sequences, such as sequencing methods, PCR methods such as real time PCR, TaqMan methods, mass spectrometry, Temperature Gradient Gel Electrophoresis (TGGE), Denaturating Gradient Gel Electrophoresis (DGGE) and genotyping methods, such as mass spectrometry based genotyping, wherein said methods are optionally coupled such as SBE-PCR followed by mass spectrometry and optionally comprise a purification step.
8. Method according to any of the preceding claims wherein the amplified sequences are purified prior to the determination of their amounts via methods selected from the group of methods comprising gel filtration, DNA precipitation and PCR product purification.
9. Method according to clam 8 wherein the amplified sequences are purified by gel filtration using an agarose gel followed by band excision and gel extraction.
10. Method according to any of the preceding claims wherein said unique sequence in the cDNA and said unique sequence in the genomic DNA are present in equal copies in the corresponding DNA.
11. Method according to claim 10 wherein said unique sequence in the cDNA is present only once in the cDNA and said unique sequence in the genomic DNA is present only once in the genomic DNA.
12. Method according to claims 1 to 9 wherein said unique sequence in the cDNA and said unique sequence in the genomic DNA are present in different copies in the corresponding DNA and said factor is taken into account when determining the amounts in step e).
13. Method according to claims 10 to 12 wherein the copy numbers of the unique sequences are determined by performing PCR reactions using either purified cDNA or purified genomic DNA as template, in each case over a concentration range of the corresponding DNA with fixed primer concentrations.
14. Method of designing primers which can be used according to the method of claim 1 comprising at least the steps of:
Identifying a gene which is stably expressed from the genomic DNA the cDNA originates from, such that the coding sequence of said gene is present in the cDNA;
Screening said coding sequence in the cDNA and the total genomic DNA for a sequence which is substantially equal regarding its composition and length but differs in at least one base; Designing a forward primer covering about 20 bases in 5' to 3' direction at the 5 ' end of said sequence which is substantially equal;
Designing a reverse primer covering about 20 bases in 5 ' to 3 ' direction on the complementary strand at the 3' end of said sequence which is substantially equal.
15. Method according to claim 1 wherein the primers designed according to claim 14 are in 5' to 3' direction a forward primer of the sequence GAGCACAGAGCCTCGCCT and a reverse primer of the sequence TCGTCGCCCACATAGGAA.
16. Use of a method according claims 1 to 15 to determine the amounts of cDNA and genomic DNA in a sample.
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