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WO1998013521A1 - Procede pour l'analyse differentielle de l'expression des genes par la reaction en chaine de la polymerase par transcription inverse primee aleatoire - Google Patents

Procede pour l'analyse differentielle de l'expression des genes par la reaction en chaine de la polymerase par transcription inverse primee aleatoire Download PDF

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Publication number
WO1998013521A1
WO1998013521A1 PCT/EP1997/005290 EP9705290W WO9813521A1 WO 1998013521 A1 WO1998013521 A1 WO 1998013521A1 EP 9705290 W EP9705290 W EP 9705290W WO 9813521 A1 WO9813521 A1 WO 9813521A1
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Prior art keywords
primers
pcr
primer
sequence
sequences
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Giangiacomo Consalez
Riccardo Fesce
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Ospedale San Raffaele SRL
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Ospedale San Raffaele SRL
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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

Definitions

  • the present invention concerns a method for the differential screening of gene expression by random primed Reverse Transcription- Polymerase Chain Reaction (RT-PCR) and a kit to be used for the performance of said method.
  • RT-PCR random primed Reverse Transcription- Polymerase Chain Reaction
  • differential gene transcription focusses on molecular mechanisms involved in major biological processes, such as cell differentiation, cell division, embryonic development and neoplastic transformation.
  • a multitude of techniques has become available in recent times to isolate differentially expressed genes. These techniques can be grouped in two classes: subtractive hybridization and differential screening. Hybridization- based differential screening and subtractive techniques are extensively covered elsewhere (1) .
  • DD Differential Display
  • cDNAs are synthesized by means of anchored oligo-dT primers to select subsets within given rnRNA populations.
  • First strand cDNAs are subsequently PCR-amplified using the same downstream oligo-dT primer and an upstream random decamer.
  • the complex PCR product is separated through a polyacrylamide gel and detected by autoradiography thanks to the incorporation in the PCR reaction of a radioactive dNTP .
  • the technique aims at pinpointing bands corresponding to differentially expressed genes from a background of ubiquitous, constitutively expressed products.
  • RNA fingerprinting protocol was developed by other authors (6) , to permit internally primed PCR amplification of oligo-dT- primed or random-primed cDNAs .
  • RAP-PCR only arbitrary primers are used for the radioactive PCR amplification step.
  • GGC unpublished observations
  • RNA fingerprinting has not been systematized to maximize coverage of genes expressed in a given tissue or cell line within a discrete number of PCR amplifications.
  • the main problem with RAP PCR lies within the unavailability of a rationally designed panel of primers permitting an exhaustive, nonredundant survey of gene expression in a given biological system.
  • the present invention solves the above problem by means of a computer-assisted search for RNA fingerprinting primers characterized by high amplification efficiencies and a marked, nonrando affinity for coding regions.
  • the collection of reagents generated according to the invention allow the use of internally primed, PCR-based RNA fingerprinting as a reasonably simple, exhaustive and systematic tool for the analysis of differential gene expression, and as a workable, advantageous alternative to differential display.
  • the method of the invention is characterized in that the PCR is carried out using a plurality of oligonucleotide primers the sequence of which has been determined by a method comprising the following steps: a) generation of random primer sequences having a CG/AT ratio of 2:1, no stop codon, no more than three consecutive identical nucleotides and no palindromic 5' and 3 ' ends ; b) screening of the primer sequences generated in a) by simulating PCR reactions on non-redundant mammalian nucleotide sequence databank entries containing at least 1,000 bp of coding region and calculating for each primer sequence their: V
  • efficiency index said efficiency index being defined as the ratio of the number of PCR products comprising coding sequences obtained using said primer sequence to the modal number of PCR products comprising coding sequences obtained for each of the whole set of tested primers generated in a) ; and (ii) selectivity index, said selectivity index being defined as the ratio between the probabilities of yielding a PCR product comprising coding sequences or 3' untranslated regions; and c) selecting some or all of the primer sequences screened in b) according to their efficiency index and selectivity index for use in PCR.
  • the invention also provides a kit for differential screening of gene expression in biological samples by means of random priming RT-PCT comprising: a) a plurality of oligonucleotide primers selected according to the above described method; b) reagents for the reverse transcription and amplification reactions; c) optionally, protocols for the cloning of the products of differential screening.
  • the primers selected according to the criteria of the claimed method allow the detection of more than 80% of cDNAs containing significant portions of coding regions, compared with about only 10% of cloned products containing translated regions obtainable according to the prior art methods.
  • the present invention provides therefore a useful tool allowing easier recognition of new sequences as well as an easier comparison between known genes and the new genes cloned by the method of the invention.
  • Figure 1 Histogram of the number of simulated PCR products (in CDS) per primer, tested on human nonredundant pseudo- cDNA database. Also shown is the expected distribution of number of products per primer, based on the probability of matching after randomly scrambling the sequences in the database (dashed line) .
  • Figure 2 Scatter plot of the number of simulated CDS PCR products yielded by each primer when tested on the human (abscissa) or mouse (ordinate) pseudo-cDNA databases (454 primers) .
  • Figure 3 Exhaustivity and redundance analysis on simulated PCR (96 most efficient primers tested on human nonredundant pseudo-cDNA database) .
  • Panel A shows the distribution of the number of simulated PCR products per transcript.
  • Solid line expected distribution, based on the probability of matching to the randomly scrambled sequences in the database.
  • Dashed line expected distribution for an increase in theoretical probability of matching by a factor equal to the ratio observed/expected mean number of products per transcript .
  • Panel B shows the distribution of the number of different primers yielding simulated PCR products from each transcript. Dashed line: expected distribution after correcting the theoretical matching probability as in panel A.
  • Figure 6 shows sample gels obtained through computer-driven fingerprinting (RF) experiments. Uninduced Hep G2 cells and some line induced with reducing agents are compared. RNA extractions, RT reactions and PCRs are done in duplicate. Ul, U2 : uninduced; 11, 12: induced. Detailed Description of the Invention
  • the random sequences described in a) of the above method can be generated easily using straight-forward computer algorithms.
  • Simulation of PCR reactions as in b) of the above method can be performed by, for example, searching both strands of the target sequence for a sequence complementary to the primer sequence, permitting varying degrees of mismatch, for example 3 mismatches.
  • a PCR product is scored if a suitable match is found on both strands and the matching sequences are within a predetermined distance from each other, for example from 100 to 1,000 bp apart. Searches can be performed using using any of several commercially available software packages, such as FINDPATTERNS in the Wisconsin GCG package.
  • Non-redundant nucleotide sequence databases are used to provide target sequences for PCR simulations.
  • Nucleotide sequence databases are easily accessible to skilled person, two of the largest and most well-known being the Genbank and EMBL databanks. From these databanks, a subset of sequences are selected. Only sequences containing at least 500 bp, preferably at least 1000 bp, of coding sequence are selected. Furthermore redundant sequences are eliminated. That is to say, for any given gene, often more than one entry occurs in the databank and it is desirable to select only one of the entries if they all have very similar sequences.
  • One method of achieving this is to compare databank entries which have a common word in their sequence descriptions with a sequence comparison program, for example FASTA, and eliminate the shorter sequence if the two sequences have sequence identity above a percentage threshold, preferably greater than 95%. It is also d desirable to eliminate intron sequences from genomic sequences to produce a contiguous cDNA sequence.
  • a sequence comparison program for example FASTA
  • the oligonucleotide primers may be of any length and preferably comprise from at least 10 to 20 nucleotides, for example they may consist of 12, 15 or 18 nucleotides, more preferably 12 nucleotides. Primers may contain additional groups such as labelling groups, for example biotin, or radio- labelled substituents . Primers may be synthesised using standard methods known to those skilled in the art, for example using an automated oligonucleotide synthesiser.
  • the efficiency index as defined above and used as one of the two criteria for selecting candidate primers should not be either too low (preferably > 2) or too high (preferably ⁇ . 10) .
  • the selectivity index as defined above and used as the other criterion for selecting candidate primers is preferably higher than 1, more preferably higher than 1.8, even more preferably higher than 2.
  • Some primers may produce PCR products containing both coding sequences and untranslated regions. However, the amount of coding sequence in some cases may be very small. Therefore, when determining efficiency and selectivity scores it may be preferable to only consider a primer as having yielded a product within a coding sequence if the amount of coding sequence within the product exceeds a predetermined percentage, for example 10, 30, 50 or 70%, or a predetermined length (e.g. 50, 100 or 200 nts) .
  • a predetermined percentage for example 10, 30, 50 or 70%, or a predetermined length (e.g. 50, 100 or 200 nts) .
  • the set of primers selected according to the method of the invention described above may be further selected from to produce a smaller set of primers. This can be accomplished by simply selecting primers with the highest selectivity indices. For example, if 500 primers are selected after steps a) to c) , it would not be necessary to synthesise all 500 primers for use in PCR techniques. A skilled person may only select, for example, from 10 to 100 primers. Generally, a skilled person would select primers with the highest selectivity index and efficiency index except that they may discard any primers with sequences that are too similar to other selected primers, for example if they are greater than 80% identical overall (or greater than 60% identical in the last 8 nucleotides at the 3 ' end) .
  • the kit described above will typically contain about from 10 to 200 primers, or pairs of primers degenerate at one position (e.g. the last nucleotide at the 3' end) preferably from 20 to 100 primers (or pairs) , for example 30, 60 or 96 primers (or pairs), selected by the method of the invention.
  • PCR simulations were run on two nonredundant (nr) databases, obtained from a combination of human or mouse sequences deposited into the Genbank and EMBL nucleotide sequence databanks (accessed through the GCG Wisconsin package, version 8.1-UNIX, August 1995) (7), using one arbitrary 12 -nt primer sequence at a time, thus assuming to each primer to anneal in a degenerate fashion to the sense and antisense strand.
  • the reduced human and mouse databases were obtained by selecting human or mouse sequences containing at least 1000 bp of coding region (CDS) .
  • CDS coding region
  • variable regions of immunoglobulins and T-cell receptors were eliminated, and all pairs of sequences sharing a word in their product descriptions were compared by the FASTA algorithm (8) ; the shorter one was eliminated when >95% identical to the other.
  • Intronic regions were eliminated from genomic sequences, generating new transcribed sequence files containing uninterrupted cDNA.
  • Annealing of the primers was simulated by searching both strands for the sequence of each primer in the nr databases by means of the FINDPATTERNS program in the Wisconsin GCG package (7) , permitting a maximum of 3 mismatches. All pairings with one or more mismatched base (s) among the last 4 (at the 3 ' end) were excluded as unsuitable to prime a polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • simulated PCR products were tagged with a CDS flag, if they contained a coding sequence portion, a UTR flag, if they contained a portion of 3' untranslated region.
  • Each primer could be assigned an "efficiency” score (total number of simulated PCR products in the sequence database) and a “selectivity” score (ratio of the probabilities of yielding a PCR product comprising // coding sequences or untranslated -3' regions) .
  • a crucial aspect in assessing the validity of the approach proposed here is to exclude the possibility that differences in "efficiency" observed among random primers are due to chance.
  • S s is a sequence composed of a s C/G nucleotides and b s A/T nucleotides
  • the probability of a G or C nucleotide in the primer matching an arbitrary nucleotide in the sequence is
  • P s A s • B s .
  • the average value of F s was 0.53 ( ⁇ 0.082) and in general P s was about 1-2-10 "4 , its value increasing for primers with increasing numbers of C/G nucleotides in the last 4 positions.
  • the ch.f. is computed by summing the logs of the single ch . f . ' s over the set of primers for the same sequence) .
  • a third distribution of interest is that of the number of "successful" primers per sequence (i.e. yielding at least one PCR product from the sequence), P3. This is computed in the same way using the modified p.d.f.,
  • Distribution P ⁇ is used to check whether the observed distribution of PCR products per primer significantly departs from the expectation: if a marked excess of particularly "good” and “poor” primers are found, this argues against a purely random distribution of nucleotides in the sequences of the databank.
  • Distributions P2 and P3 yield information on the exhaustivity of the approach, i.e. the capability of picking out as many different sequences as possible.
  • the shape of the p.d.f. P3 can be compared to the corresponding distribution, obtained by the simulation experiments, to check whether any bias is present towards a subpopulation of sequences (i.e. whether some sequences are significantly more subject to amplification than others) .
  • Reverse transcription is carried out using a (dT) 16 primer on 1 mg total RNA extracted by the caesium chloride method (9) .
  • Radioactive PCR reactions in duplicate, are performed from 2 ⁇ l of each RT reaction in 50 ⁇ l final volume with arbitrary 12-mers (final cone. 4 m ) , using Perkin Elmer 1 x Amplitaq polymerase + MgCl2 [1.5 . mM] .
  • PCR conditions are 3 minutes at 94 °C, 2 minutes at 80°C at which /
  • Taq polymerase is added (hot start) , followed by 35 cycles of 40 sees, at 94°C, 1 min. at 50°C, 1 min. at 72°C, with a final elongation step of 5 ins at 72°C.
  • 0.2 ⁇ l [ ⁇ - 32 P]dCTP are added to each reaction.
  • Amplified products are separated on a 5% denaturing polyacrylamide gel and visualized by autoradiography . Differentially displayed bands are cut from the gel and electroeluted in dialysis bags as described (2) . Bands are reamplified using the same 12-mer primers and cloned into a modified pBluescript II SK+ (Stratagene) . Clones corresponding to differentially displayed bands are selected from the background of unrelated products.
  • a series of 1000 acceptable primers (according to criteria a, b and c) were challenged against the human nr database to measure their efficiency (total number of simulated PCR products) and selective affinity for coding portions of transcripts. Only primers yielding >100 simulated PCR products out of 2 , 085-sequences of our human nr database (6.04 Mb total DNA, 72.8% CDS) were included in the "good primers" list (158 primers) ,- 497 primers were discarded because of their similarity to previously included primers (criterium d) . The remaining 345 primers yielded ⁇ 100 simulated PCR products.
  • Figure 1 illustrates a histogram of the number of simulated PCR products obtained from each primer in this series (503 primers) . Also illustrated is the probability density function (%) expected based on the probability of matching to the randomly scrambled database sequences (see Section b for details on the computation of this curve) . It can be clearly seen that the observed distribution does not fit a random distribution of bases in the sequences, and that the extreme shoulders of the distribution curve are markedly overcrowded. This indicates a large excess of particularly poor and particularly efficient primers, and points to the IS possibility of selecting efficient PCR primers based on the present "simulated gene fishing" approach.
  • the simulation was repeated (MOUSE12.8) by testing the same series of primers on the mouse nr nucleotide database, containing 1041 sequences comprised of 2.95 Mb total nucleotide sequence, (72.5% CDS), in order to check whether species-specific features in sequence composition played a major role in determining the efficiency scores of different primers. In this case 193 "good" primers and 326 inefficient ones were obtained; 481 primers were not considered due to criterium d. The distribution of the numbers of simulated PCR products per primer was qualitatively similar to the one obtained in the human database (not shown) .
  • Figure 2 is a scatter plot of the number of simulated PCR products obtained in the human (x-axis) vs. the mouse (y- axis) database, with the same primers (454) .
  • some "genetic strings" the base composition of some particular oligonucleotides
  • the primers selected because of their ⁇ c high efficiency in yielding simulated PCR products may be directed towards subpopulations of genetic sequences. If this were the case, the number of sequences not picked out
  • Figure 3 illustrates this point; in particular, it shows the distributions of the numbers of PCR products generated by each primer in the human database (A) , and the distribution of the numbers of different primers yielding at least one PCR product from each transcript (B) .
  • Data are relative to sequences containing at least 1000 bp of coding region.
  • the dashed line in B represents the expected distribution of numbers of primers (among the 96 selected here) picking out each sequence, given the average value of such distribution
  • Figure 4 shows that the best 96 primer pairs yield a distribution of number of primers spotting each sequence which is shifted to the right with respect to the expectation (solid line) , suggesting that this subset of primers is particularly efficient; the distribution is well fit when the expectation is corrected for the mean efficiency of this particular set of primers (dashed line) and the number of sequences yielding no simulated PCR products, d) Experimental assessment of the primer panel
  • Sensitivity Besides assessing the above issues, we tried to determine whether our internal primers exhibit a preference for highly abundant transcripts (sensitivity) in at least two cases, cDNA clones generated by our method produced no hybridization signal by northern analysis of 30 mg total RNA. In two cases, cDNAs found by RT-PCR in a given tissue have required plating of over 10 ⁇ pfu to permit isolation of the corresponding gene by hybridization-based screening of the appropriate library.

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Abstract

La présente invention concerne un procédé pour l'analyse différentielle de l'expression de gènes par la réaction en chaîne de la polymérase par transcription inverse primée aléatoire. L'invention concerne aussi un ensemble pour mettre en oeuvre ce procédé.
PCT/EP1997/005290 1996-09-27 1997-09-26 Procede pour l'analyse differentielle de l'expression des genes par la reaction en chaine de la polymerase par transcription inverse primee aleatoire Ceased WO1998013521A1 (fr)

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EP97911157A EP0941362A1 (fr) 1996-09-27 1997-09-26 Procede pour l'analyse differentielle de l'expression des genes par la reaction en chaine de la polymerase par transcription inverse primee aleatoire

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GB9620216.3 1996-09-27
GB9620216A GB2319082A (en) 1996-09-27 1996-09-27 Differential screening of gene expression by random primed reverse transcription - polymerase chain reaction (RP RT-PCR)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6900053B2 (en) * 2001-09-14 2005-05-31 Isis Pharmaceuticals, Inc. Antisense modulation of fibroblast growth factor receptor 2 expression
WO2014081323A2 (fr) 2012-11-22 2014-05-30 Uniwersytet Jagielloński Procédé d'identification de virus à arn et son application
CN120126558A (zh) * 2025-02-26 2025-06-10 广州金墁利医药科技有限公司 一种模拟引物在基因组上扩增子的方法、装置、设备及存储介质

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995033760A1 (fr) * 1994-06-06 1995-12-14 Brigham & Women's Hospital Procede pour conduire des phases d'hybridation d'acide nucleique sequentielles

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995033760A1 (fr) * 1994-06-06 1995-12-14 Brigham & Women's Hospital Procede pour conduire des phases d'hybridation d'acide nucleique sequentielles

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AYALA M ET AL: "NEW PRIMER STRATEGY IMPROVES PRECISION OF DIFFERENTIAL DISPLAY", BIOTECHNIQUES, vol. 18, no. 5, 1995, pages 842 - 850, XP002033062 *
BAUER ET AL.: "IDENTIFICATION OF DIFFERENTIALLY EXPRESSED mRNA SPECIES BY AN IMPROVED DISPLAY TECHNIQUE (DDRT-PCR)", NUCLEIC ACIDS RESEARCH, vol. 21, no. 18, 1993, pages 4272 - 4280, XP000394394 *
LINSKENS M H K ET AL: "CATALOGING ALTERED GENE EXPRESSION IN YOUNG AND SENESCENT CELLS USING ENHANCED DIFFERENTIAL DISPLAY", NUCLEIC ACIDS RESEARCH, vol. 23, no. 16, 1995, pages 3244 - 3251, XP002047039 *
WELSH J ET AL: "ARBITRARILY PRIMED PCR FINGERPRINTING OF RNA", NUCLEIC ACIDS RESEARCH, vol. 20, no. 19, 11 October 1992 (1992-10-11), pages 4965 - 4970, XP000508271 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6900053B2 (en) * 2001-09-14 2005-05-31 Isis Pharmaceuticals, Inc. Antisense modulation of fibroblast growth factor receptor 2 expression
WO2014081323A2 (fr) 2012-11-22 2014-05-30 Uniwersytet Jagielloński Procédé d'identification de virus à arn et son application
CN120126558A (zh) * 2025-02-26 2025-06-10 广州金墁利医药科技有限公司 一种模拟引物在基因组上扩增子的方法、装置、设备及存储介质

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EP0941362A1 (fr) 1999-09-15
GB9620216D0 (en) 1996-11-13

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