US20070065830A1

US20070065830A1 - Cloning multiple control sequences into chromosomes or into artificial centromeres

Info

Publication number: US20070065830A1
Application number: US11/074,265
Authority: US
Inventors: Roger Lebo; James Malone
Original assignee: Childrens Hospital Medical Center of Akron
Current assignee: Childrens Hospital Medical Center of Akron
Priority date: 2002-09-04
Filing date: 2005-03-07
Publication date: 2007-03-22
Also published as: US8548747B2; US20060031052A1; US20090155794A1; US20060031051A1

Abstract

Artificially synthesizing 29 homozygous cystic fibrosis core panel controls demonstrates placing multiple homozygous mutant sequences on the same single control DNA sequence to streamline quality control by minimizing extra control assays, time, and costly formatted test materials and testing all controls during every test. Any rare or unavailable reported DNA sequence can be PCR amplified using primer pairs synthesized with the designated mutation or variant sequence with paired adjacent upstream and downstream primers to amplify target sequences in total genomic DNA.

Description

SUMMARY

When tested together, twenty nine artificially synthesized, cloned, homozygous cystic fibrosis controls streamline test quality control by minimizing control assay number, cost, and assay time. Any rare or unavailable reported sequence can be PCR amplified from total genomic DNA or RNA template using a synthesized primer incorporating the mutant, polymorphic or variant sequence and a paired upstream or downstream primer. These synthesized upstream and downstream fragments overlap at the modified site and are amplified together to obtain a homozygous mutant control. The 5′ and 3′ flanking sequences were selected to span all the PCR sites in the test platforms we were using. In this manner 1 to 4 homozygous mutations were artificially synthesized into each of 17 fragments 433 bp-933 bp long by PCR amplification on total normal human genomic DNA template. Then one to three synthesized fragments were blunt end cloned into 9 vectors and validated by sequencing. Together these mutations included (1) 7 mutations on two joined fragments cloned into a single vector's multiple cloning site and (2) all 25 homozygous mutations originally recommended by ACMG for the core cystic fibrosis panel which are sufficiently long to be amplified by the PCR primer pairs in most commercial platforms. Together these cloned mutant sequences provide controls for each PCR assay to optimize multiplex test reliability for all recommended ACMG mutations.
Key words: controls: homozygous, synthetic, PCR amplified, multiplex
Achieving a reliability of 99% for any 25 mutation test requires an individual mutation test reliability of 99.96% (1 incorrect result among 2500 reported results). In contrast, achieving a test reliability of 99% for a 100 mutation test requires an individual mutation test reliability of 99.99% (1 incorrect among 10,000 reported results). When applying ever larger multiplex tests, simultaneously testing controls for all target sequences is required to maintain the most reliable molecular genetic analyses. Thus the College of American Pathologists' Molecular Genetics Committee dictates that whenever possible a control needs to be included in an independent control reaction for each molecular assay and that the best available control is to be used. At the same time, obtaining controls for each reported gene and its common mutations is typically the most difficult hurdle required to introduce any new assay into a Clinical Molecular Genetics laboratory's test menu. The following protocol demonstrates the relative ease required to synthesize control sequences of standard PCR amplifiable lengths for use by multiple laboratory specific and commercial multiplex cystic fibrosis test platforms.
The cystic fibrosis transmembrane receptor (CFTR) gene is mutated in both alleles in patients with cystic fibrosis (CF [MIM 21977]) and congenital bilateral aplasia of vas deferens (CBAVD [MIM 277180]). Cystic fibrosis is the most common lethal genetic disease in Caucasians, affecting about 1 in 2500 newborns while CBAVD renders about 1 in 5,000 males sterile with about half of these patients exhibiting cystic-fibrosis like symptoms and a few have full-blown cystic fibrosis. Over 1000 mutations and 50 polymorphisms have been reported worldwide throughout a large portion of this 24 exon CFTR gene with its 4600 basepair coding sequence that spans 188 kb of genomic DNA (Cystic Fibrosis Mutation Database.)
The ACMG 25 mutation cystic fibrosis panel was selected by the Cystic Fibrosis Committee of the American College of Medical Genetics for screening pregnant Caucasian women who are at a more substantial risk of having a cystic fibrosis fetus than women from other races (Grody et al. 2001). These selected 25 CFTR mutations included all mutations with a frequency of at least 0.1% in cystic fibrosis patients. This was considered an attainable goal for a large number of laboratories to offer routinely and at the same time charge a reasonable fee to encourage third party payment for their laboratory service. Currently thousands of pregnant Caucasian patients are tested each month by dozens of laboratories as a first step in detecting most fetuses at-risk for cystic fibrosis.
Although initially developed for population carrier screening, the 25 mutation core panel can also be used to screen patients with suspicious symptoms because over 90% of all affected Caucasian patients will test positive for at least one mutation including over 99% of affected Northern European Caucasian patients (Lebo and Grody, unpublished data). We incorporated the 5T allele into the select group of 25 most common mutations tested in symptomatic patients because (1) the 5T allele with the severe ΔF508 mutation and the 5T allele with decreased penetrance are the two most common alleles resulting in cystic fibrosis-like symptoms including CBAVD, (2) 0.17% to 3.4% of cystic fibrosis patients with severe symptoms have one allele with the 5T sequence without any other detected mutation, and (3) about 40% of CBAVD patients are compound heterozygotes for 5T and another CFTR gene mutation (Claustres et al. 2000; Kerem et al. 1997; Lebo and Grody, unpublished data).
When the 25 mutation panel was selected by the ACMG Cystic Fibrosis Committee, the controls for each of these mutations were unavailable in any one location. Thus many laboratories expanded their CFTR mutation testing panel to 25 mutations and began offering the service without simultaneously testing all the appropriate controls. In order to meet the substantial new demand for better controls, Coriell Cell Repository, Camden, N.J., aggressively collected, transformed, and distributed human cell lines that contained all available mutations among those to be tested. In spite of substantial efforts to date, Coriell has yet to market a collection of cell lines that together contain all 25 mutations. Coriell has been marketing total genomic DNA from cell lines transformed with Epstein Barr virus with most of these represented as heterozygous mutations as a Product of Substantial Equivalence. Genzyme, which began testing about 87 mutations a decade ago, had to complete many thousands of tests over several years to identify and then incorporate a patient DNA control for each of the 87 selected mutations. Furthermore, testing all 25 cystic fibrosis mutations in the Coriell collection with each unknown set of patient samples requires a substantial investment in labor and test materials. For this reason dozens of laboratories doing 25 mutation tests have been rotating selected control patient DNAs from Coriell through their regular clinical protocol. In this fashion, all available mutation controls are only tested among several independent multiplex assays run on different days, but all mutant sequences are never tested at the same time in a single multiplex assay. Furthermore, most of the Coriell Cell lines are heterozygous at the CFTR mutation site so that these controls cannot be mixed together because the proportion of normal alleles increases each time another heterozygous genome is introduced. Therefore these heterozygous controls must be used individually and cannot be used to determine whether an assay distinguishes between a homozygous and heterozygous DNA sample, the primary deficiency in our studies (See Discussion).
No matter how rare or unavailable, all selected reported mutations, polymorphisms, and variants can be synthesized not only as homozygous but also as heterozygous controls using PCR primer pairs with mutant sequences, genomic DNA template, and editing DNA polymerase. Prior to offering a 25 cystic fibrosis mutation test at Akron Children's Hospital three years ago, our laboratory artificially synthesized and verified homozygous controls for all reported mutations by independent assays on the Innogenetics multiplex format (FIG. 5). This strategy reduced the energy and materials required to assay greater than a dozen control reactions with permanent Coriell cell line DNAs to three Innogenetics PCR amplification reactions and two ASR detection strips (Lebo et al. 2003). Alternatively, mixing all homozygous mutations together resulted in a single multiplex control for the TM Biosciences platform (FIG. 6). Validation of these controls on other platforms was addressed by another study completed by Acrometrix Corp. (Benicia, Calif.; Aytay et al., 2005).

MATERIAL AND METHODS

Primers were selected incorporating published mutations into the mutation-specific primers and selecting the background sequences including the flanking primers from the CFTR gene sequences in The Genome Database. All primers were synthesized by Invitrogen. Pfu DNA Polymerase or Pfu Ultra High-Fidelity DNA Polymerase was used for high fidelity PCR amplification (Stratagene, La Jolla, Calif.). All PCR amplifications were performed according to recommended assay conditions and protocols for the selected enzyme (FIG. 1-III,V,VII,IX; See Results, for Synthetic Strategy). The PCR amplified products were sized by fluorescent ethidium bromide observation following agarose gel electrophoresis. Many PCR reactions produced a single fragment of the expected length, but when other fragments were also amplified, the appropriate fragment was purified by preparative agarose gel electrophoresis prior to subsequent PCR amplification or cloning. The purified amplified fragments were quantified and blunt end cloned into competent bacteria. After growth and purification, the plasmid DNA digested with appropriate restriction enzyme(s) was electrophoresed to confirm that the insert had remained the correct length. The clones were independently validated by testing with one or two commercial cystic fibrosis test kits (Innogenetics, TM Biosciences; FIGS. 5,6) and by bidirectionally sequencing the cloned insert and its flanking vector sequences (the Gold Standard for Clinical Molecular Genetics
Results
PCR Amplification with Synthesized Mutant Primers
Our goal was to design and synthesize a complete set of ACMG-recommended homozygous CFTR gene mutation controls for use in the widest variety of commercial and laboratory-specific CFTR platforms. Thus pairs of PCR primer sites were selected to span all known primer sites reported to amplify each CFTR mutation-containing region (The Genome Database; FIG. 1-I, F1 and R2). Then forward and reverse primers were selected to span and incorporate one or more mutations between the flanking primer sites (FIG. 1-I, primers F2 and R1 with M1 and M2 mutation sites). The primers were selected, synthesized, and used for PCR amplification of normal genomic DNA template with primer pair F1 and R1 synthesizing the mutation-containing upstream Product #1 (FIG. 1-II) and primer pair F2 and R2 synthesizing the mutation-containing downstream Product #2 (FIG. 1-III). Then the flanking forward and reverse primers F1 and R2 (FIG. 1-III) were added to aliquots of Products #1 and #2 which were spliced together in a unique orientation by PCR amplification to give Product #3 flanked by F1 and R2 sequences with the M1 and M2 mutations in the middle (FIG. I-IV).
Additional mutations were added to a synthesized mutation-carrying fragment when another homozygous mutation was desired on the same gene fragment. For example, when mutation M3 was desired on the same fragment as mutations M1/M2, it was synthesized onto Product #3 template using forward F3 and reverse R3 primers synthesized to include mutant sequence M3 (FIG. 1-V to 1-VII). Analogous to the protocol to add the M1/M2 mutations above, upstream Product #4 and downstream Product #5 were synthesized using Product #3 as template prior to splicing these together into Product #6 using flanking primers F1 and R2. Product #6 now contains mutations M3 and M1/M2. The same protocol can be used to incorporate another homozygous mutation or substitute a new mutation for an existing mutation on Product #6 (FIG. 1,V-VIII).
Initially 31 of the first 34 manually selected primer pairs amplified the correct target sequences. Three flanking primer sites that initially failed to amplify a correct length unique product were moved to an even more distant location from the mutation site(s), thus assuring that the synthesized mutation-carrying CFTR fragment would also span all the known primer sites reported to amplify the gene region to be tested. All 3 additional selected primers amplified the site for which each was designed.
Our design included a plan to synthesize a small number of fragments containing all 29 selected CFTR mutations that would provide an optimal multiplex control according to the selected commercial or private format using the minimal number of control mixtures. The number of mutations that can be added to a single sequence is determined by the minimal distance between mutations required to distinguish unambiguously between normal and mutant sequences tested by the selected assay format including (1) nitrocellulose filters with slot blot designated locations, fluorescent beads, or microchips each with locations to which ASOs are hybridized uniquely, or (2) Mass Spec that hybridizes complementary nucleotide sequences adjacent to the mutations to be measured and then adds additional 3′ nucleotides until a base complementary to the single base subtracted from the reaction mixture is encountered. For instance, all our homozygous controls are tested with each assay to assure that each control unambiguously gives a homozygous mutant signal with no cross hybridization to the normal sequence.
When heterozygous controls are desired for applications like Mass Spec, multiple approaches can be used. For instance, normal genomic sequences can be PCR amplified directly from total normal genomic template DNA using the flanking primers like F1 and R2 (FIG. 1,I-IV). These amplified normal sequences can then be checked for size by electrophoresis, cloned, and each sequence verified by bidirectional sequence analysis through and beyond the vector's cloning site. Then these normal cloned normal gene sequences can be added to the cloned homozygous synthetic controls in equal copy number. Alternatively, Products #3A and #4A can be PCR amplified together typically in equal concentrations using forward primer F3 for the upstream mutation M3, reverse primer R1 for the M1/M2 mutations, and F1 and R2 for the flanking primers (FIG. 1,XI-XIV; Lebo et al., 2000).
The number of homozygous mutations that can be added to any single DNA fragment that can be tested unambiguously depends upon the assay approach used by the selected CFTR test platform as well as the proximity of each mutations pair, the type and size of mutation (nucleotide substitution(s), insertion, or deletion), and the immediately flanking sequences of each mutation. For instance, the Intron 10/Exon 11 fragment spans 5 common mutation sites: 1717-1G->A, G542X, G551D, R553X, and R560T (FIG. 2) while the ΔF507 and ΔF508 mutations in Exon 10 overlap by 1 basepair and each delete three basepairs. Thus the F507 and ΔF508 mutations which overlap by 1 basepair were synthesized and cloned independently. Because the G551D and R553X mutations are within 4 basepairs, these mutations were also synthesized on independently cloned Intron 10/Exon 11 fragments that each carried three other mutations: 1717-1G->A, G542X and R560T. This design provided a means to mix these mutant sequences differently to optimize control amplification and detection reaction number for both the commercial Innogenetics and TmBiosciences CFTR multiplex platforms (See below).
Following these strategies, 17 mutation-carrying fragments were synthesized and tested with the Innogenetics assay to confirm that these fragments carried the expected mutations. Some of the PCR fragment primers were designed to overlap with more than one independent fragment to allow pairs of amplicons to be spliced together in the orientation of choice by PCR. Then the 17 fragments were spliced together into 9 fragments and blunt end cloned. For instance, Product #9 (FIG. 1,X) was synthesized using the same approach as Product #3 (FIG. 1,I-IV) except that the upstream primer R2′F4 was selected in place of upstream primer F4. Then synthesized Product #9 is spliced to Product #6 by mixing and amplifying aliquots of these products together using flanking primers F1 and R5 to synthesize Product #10 with mutations M1, M2, M3, and M4 (FIG. 1,IX-X). Additional mutation-containing CFTR segments can be spliced to Product #10 in this manner. Thus one PCR product was synthesized from 3 joined fragments by repeating the splicing reaction to add a third mutation-carrying amplicon. In this fashion, the first 29 PCR amplified mutations on 17 synthesized fragments some of which were spliced together to form 9 fragments (FIG. 2).
Following the final PCR amplification and purification when required, the 9 spliced fragments were inserted into the cloning site of a single vector. Inserts isolated from individual clones were screened for the correct size after restriction enzyme digestion. Then the presence of the synthesized CFTR mutation(s) and flanking sequences were detected using the Innogenetics CFTR multiplex mutation assay and further analyzed by bidirectional sequencing of mini-prepped plasmid DNA. A 30% aliquot of a single miniprepped sample isolated from a 3 ml suspension culture provided substantially more than sufficient material for bidirectional sequencing, validating two commercial platforms, and providing complete controls for each of the thousands of cystic fibrosis samples tested at Akron Children's Hospital (Lebo et al. 2003). Bidirectional sequencing was completed by Cleveland Genomics (Cleveland, Ohio) using M13 sequencing primers hybridizing to these sites in the cloning vector and selected internal PCR primers used in the original PCR synthesis. Each of the 9 fragments carried the expected subset of 29 different cystic fibrosis mutations (FIG. 3). One of the nine clones has an unreported 622-194G->A variation in intron 4 that is 284 basepairs removed from the 711+1G->T mutation in exon 5. Three other variations found among these 6596 cloned DNA basepairs are found in the CFTR (cystic fibrosis transmembrane receptor) database (FIG. 3).
Three Insert Clone with A455E and N1303K Homologous Regions
In 15 of 17 synthesized, cloned, and sequenced fragments amplified from total genomic DNA using synthesized DNA fragments to introduce 31 other mutant sites had only the primary CFTR sequence listed for the normal gene [CFTR Mutation Database] with the exception of three normally variant single nucleotide substitutions: 622-61A->T; 1525-61A->G, and 405+46G->T. The first clone with three inserts had to be reengineered to replace the first and third segments (FIG. 1, Bottom). The three segments were: (Left) The 5′CFTR genomic segment with A455E from exon 9 and 5T from intron 8; (Center) a segment with the 3849+10kbC->T mutation from intron 19; and (Right) the 3′ insert with the N1303K mutation from exon 21. All of the sequences in the center segment except the inserted 3849+10kbC->T site were identical to the corresponding normal CFTR genomic sequence in the CFTR mutation database. In addition, 98% of the initially amplified and cloned sequences in the 5′ A455E and 3′ N1303K containing cloned segments were also identical to the corresponding normal CFTR gene sequences in this database. However, 7 nucleotide substitutions downstream of the A455E site had been amplified from total genomic DNA that mimicked homologous sequences from the chromosome 20 pseudogene and 11 nucleotides had been substituted from a 162 bp duplication downstream of the N1303K site in intron 21 (FIG. 5). Thus additional clones from both the 3′ downstream segments from the A455E site and from the N1303K site were reselected from the transformation plate, resequenced to find one with the correct genomic sequence, and synthesized into the final clone that is being redistributed as part of the multiplex CFTR control sequences.
Validation on Two Commercial Platforms:
The Innogenetics multiplex CFTR mutation format coamplified three combinations of these 9 clones each with unique CFTR mutation combinations, and analyzed the amplified fragments on two sets of mutation-specific ASOs bound to nitrocellulose filter paper. As part of this format validation, two different Intron10/Exon11 fragments were sequenced and tested: the first with G551D along with 1717-1G->A, G542X, and R560T, and the second with R553X along with 1717-1G->A, G542X, and R560T. When tested individually, the first fragment hybridized uniquely to the G551D amplified control site as well as to the other three mutations (1717-1G->A, G542X, and R560T), but not to the normal site R553 because the G551D mutation interferes with the binding to the normal R553 sequence on the nitrocellulose filter strip (FIG. 4,f1,left). In contrast, the second fragment with the R553X mutation binds to both the R553X mutation site and also to the normal G551 site (FIG. 4,f3,right) because the R553X sequence does not prevent hybridization to the G551 oligonucleotide at the hybridization stringency and ASOs used in the assay. As illustrated, one other homozygous mutation was tested simultaneously with each of these fragments (FIG. 4,f1,left and FIG. 4,f3,right) during the development of these specific cloned sequences. We also note that the 1717-1G->A mutant site has a more intense signal than the other mutant sites G542X R553X, and R560T on the same fragment of DNA (FIG. 4,f3,right). We attribute this to different sensitivities of the assay for the PCR amplified sites.
Then all the fragments that were cloned and verified by bidirectional sequencing (FIG. 2) were mixed appropriately, amplified in three Innogenetics multiplex PCR reactions, and tested on two Innogenetics test strips. These results detected homozygous mutant signal for all the mutations tested (FIG. 4,mix,left and right). At the same time normal signals were observed for the normal 394ΔTT, and 2143ΔT sites where the normal CFTR gene sequence remained as well as the G551 site which hybridized to the normal G551 labeled ASO in spite of the adjacent R553X sequence modification. In contrast, the ΔF507 and ΔF508 mutation containing fragments were synthesized independently because these fragments could not be introduced into the same normal CFTR gene sequence at the same time. As expected, the Innogenetics ASOs analyzed both the ΔF507 and ΔF508 mutations correctly without hybridizing to the F507 and F508 normal sequence (FIG. 4,mix,right). Therefore each of the two Innogenetics test strips gave exactly the results anticipated given the sequence characteristics and the prior results obtained when the clones were analyzed individually.
In contrast, the TmBiosciences fluorescent bead assay simultaneously and unambiguously characterized all the mutant sequences even when all were mixed together into one control tube (FIG. 6). At the same time, the negative result for the R553X mutation when tested with the homozygous G551D control was 24% and the R553X mutation was 76%. All other negative normal results were less than 16%. Obviously the G551D sequence was detected by the R553 normal sequence. This is due to the test methodology used which relies on allele specific primer extension since the R553X mutation interferes with the G551 primer binding to minimize the G551 signal but give a stronger signal on the normal R553 test platform. Nevertheless, when the mechanism of this cross hybridization is understood, the single artificial mixture of the 9 cloned fragments can be tested together and interpreted unambiguously as a single TmBiosciences control reaction mixture (FIG. 6).
At the same time, these homozygous PCR amplified controls gave heterozygous signals for mutation #3 and mutation #9 for the first 9 homozygous mutations synthesized when tested on a multiplex cystic fibrosis tests from a third manufacturer. Although the manufacturer corrected the specificity of mutation #3 immediately upon learning of our results, we moved on to the Innogenetics test system when we found a second homozygous control with a heterozygous signal pattern among the first nine tested. This result emphasizes the importance of using homozygous controls over the heterozygous cell lines provided by Coriell. Obviously the third manufacturer had no way of knowing that a homozygous sample could give a heterozygous result without an appropriate homozygous control. At the same time, when less than a full panel of mutation controls is tested on any one analytical run, small changes in hybridization conditions might modify hybridization of one of the 29 targets without changing the other 28 results. Our mixed set of controls can detect both of these test specificity failures.
Discussion
Multiplex PCR amplification reactions typically cannot be relied upon to amplify each existing target site so that all can be visualized after a typical 10⁶-fold PCR amplification. For instance, one of us (RVL, unpub. results) developed a 15 site multiplex PCR test for Y chromosome deletions that was assayed in 3 groups of 5 PCR target sites. When completing 105 patient assays, 12 patients were identified and reported with deletions of one or more adjacent targets that were verified by repeat individual PCR analysis at each site that failed to amplify during the initial screen. However, another 11 of the 105 samples had individual reactions that failed to amplify existing targets in two or more sites that were physically separated by amplified sites. In these samples, the non-amplifying sites could always be amplified by repeating the initially failed PCR reaction with individual primer pairs to each previously failed site with 0.1× or 10× of the total genomic DNA originally tested or by substituting repurified genomic DNA. Thus PCR amplifying all control targets together is critical in helping to assure that multiplex PCR reactions are working effectively.
Our sequence verified controls are sufficiently long to serve as internal simultaneous multiplex PCR amplification controls as well as homozygous mutant sequence controls. Sequencing is considered the gold standard of DNA sequence validation. Our cloned, sequenced controls are nested within sufficiently long CFTR gene fragments to include all the PCR primer sites save one site in eight commercial platforms (Aytay et al, Submitted). When using heterozygous cell lines, one has not confirmed that the test system can distinguish unambiguously between homozygous and heterozygous mutations. In contrast, Bajjani and Amos have prepared multiple additional cystic fibrosis controls by synthesizing and mixing 100 bp fragments. This approach does not control for multiplex PCR amplification of homozygous mutant sites, but does require adding the control sequences to control tubes after PCR amplification, typically in a different laboratory location. Furthermore, these controls almost certainly consist of a mixture of fragments with similar but not identical sequences. When synthesizing 100 basepair control DNA fragments, the proportion of identical synthesized sequences typically drops precipitously as the synthesized length exceeds 60 basepairs. Thus the concentration of the synthetic primers can only be estimated and the reliability of 100 bp synthesized controls is anticipated to be less than perfect. Furthermore, when one requires that the most robust controls are verified by sequencing, repeated bidirectional sequencing would need to be completed prior to using any newly synthesized fragments to replace exhausted control stocks. Since readable sequence typically begins about 30-40 basepairs downstream from the end of the target, then 100 bp synthesized sequences would be verified in only 1 direction. In contrast, the entire bidirectional sequence can be determined for cloned controls by using sequencing primers some distance into the vector from the cloning site or into the adjacent cloned control fragment.
Clearly, homozygous controls for each mutant site are preferable to no control and additional mutation controls prepared by this method can be used prior to developing cloned controls. However, each desired synthesized control can be readily prepared and cloned using four selected primer sites and total normal genomic DNA that give a PCR product sufficient to control for PCR amplification. We have used these controls since we first offered a 25 mutation multiplex cystic fibrosis test in our laboratory three years ago (Lebo et al. 2003). Preparing a control that works on multiple different commercial platforms required considerably more validation time prior to offering to the general genetics community (Aytay et al., 2005).
Multiple approaches can be used to synthesize heterozygous controls. For instance, normal genomic sequences can be PCR amplified directly from total normal genomic template DNA using the same flanking primers like F1 and R2 (FIG. 1, I-IV). These amplified normal sequences can then be verified by bidirectional sequence analysis through and beyond the vector's cloning site. Then normal cloned gene sequences can be mixed with the cloned homozygous synthetic controls in equal copy number. Alternatively, Products #3 and #4 can be PCR amplified together typically in equal concentrations using forward primer F3 for the upstream mutation M3, reverse primer R1 for the M1/M2 mutations, and F1 and R2 for the flanking primers (FIG. 1,XI-XIV; Lebo et al., unpublished data). Yet another approach would be to mix existing clones of normal gene regions with cloned synthesized homozygous controls.
In summary, we have synthesized 433 bp to 933 bp sequences containing the 25 ACMG recommended mutation panel in 17 fragments inserted into 9 vector cloning sites. The entire sequences and cloning sites have been verified. These artificial mixtures can be added to test tubes adjacent to unknown samples in the PCR set up area to control for the multiplex PCR amplified cystic fibrosis test kits. Individual idiosyncrasies of test formats for which these may be employed will depend upon the precise PCR primer sites, whether these sites include normally variant sequences that interfere with PCR amplification, whether the primer pairs also amplify homologous genomic DNA sequences at other locations, and the relative amplification of each site during PCR at multiple different salt conditions found in multiple extracted DNA samples. Nevertheless, using this artificial mixture with all 25 mutations will surely improve the reliability of each cystic fibrosis assay when run as a control with every assay of unknown patient samples.
This approach provides any laboratory with a ready means to produce homozygous and heterozygous controls for any laboratory assay without requesting and sharing samples with other laboratories. When adding 32 DNA tests to the menu of the Clinical Molecular Genetics Laboratory at Boston University, my laboratory typically spent 2-4 weeks to pick, order and test PCR primers and 2-4 months to obtain the required controls from generous colleagues. Synthesizing all unavailable control DNA fragments and verifying the sequence provides a rapid means to satisfy the most stringent laboratory reviewer and further expedites test development for small groups of cooperating laboratories. Substantially larger numbers of multiplex controls can be offered through facilities with the wherewithal to develop, produce, and maintain the highest quality multiplex controls required to satisfy very large numbers of laboratories over wide geographical regions.

ELECTRONIC-DATABASE INFORMATION

Cystic Fibrosis Mutation Database, http://www genet.sickkids.on.ca/cftr/
Genetests, http://www.genetests.org/
Online Mendelian inheritance in Man (OMIM), http://www.ncbi.nlm.nih gov/OMIM/
The Genome Database, http://www.gdb.org/

REFERENCES

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FIGURE LEGENDS

1. PCR Synthesis of Homozygous and Heterozygous Controls. Sections I-X illustrates the incorporation of four defined homozygous mutations (M1, M2, M3, M4) into two different genomic fragments that are then spliced together for blunt end cloning. Sections XI-XV illustrate the incorporation of three defined heterozygous mutations (M1, M2, M3) into two different genomic fragments that when tested together give a heterozygous result. I. The first product to be synthesized has two mutations M1/M2 in a fragment with its upstream site defined by primer F1 and downstream site defined by primer R2 using total genomic Template DNA. II. PCR amplification incorporated mutations M1 and M2 into upstream PCR amplified Product #1 using reverse primer R1 containing the mutant sequences M1/M2 and forward upstream primer F1. III. In a separate reaction mutations M1 and M2 were incorporated into downstream PCR amplified Product #2 using forward primer F2 and reverse downstream flanking primer R2. Then Products #1 and #2 are mixed with additional F2 and R2 primers and amplified together to produce Product #3 (IV). V. Then mutation M3 was introduced into Product #3 using primers F1 and R3 to amplify upstream Product #4 (VI). Similarly Primers F3 and R2 amplify downstream Product #5 now with all three mutations M1, M2, and M3 (VII). Then Products #4 and #5 are spliced together with primers F1 and R2 to make Product #6 (VII) with mutations M1, M2, M3 and upstream and downstream sequences. Subsequently mutation M4 was introduced into another CFTR gene segment in an analogous fashion using M4 mutation-specific primers along with flanking primers R2′F4 and R5 to make Products #7 and #8 (not shown) and spliced product#9 (IX). Then Products #6 and #9 are spliced together by mixing these two products together and amplifying with F1 and R5 flanking primers to synthesize Product #10 (X) with mutations M1, M2, M3, and M4. These were blunt end cloned into a single vector (FIG. 2). XI. Alternatively, heterozygous products can be made using genomic template DNA, upstream and downstream primers (F1 and R2) and one primer for each site into which mutations will be synthesized. XI-XII. Product #1A with a homozygous M1M2 sequences is amplified with its own reverse primer R1 and forward primer F1. In a separate reaction homozygous Product #2A with mutation M3 is amplified using the forward M3 primer F3 and the reverse primer R2. These sites are selected so that PCR amplified Products #1A and #2A will have overlapping homologous sequences. XV-XV. Then Products #2A and #3A are mixed with forward primer F1 and reverse primer R2 and amplified to give Products #3A and #4A in the same reaction, typically in equal concentrations. In fact, PCR amplifying with primers F1, F3, R1, and R2 using total genomic template DNA also result in a mixture of Products #3A and #4A (Lebo et al. unpublished data).
2. Cloned homozygous CFTR gene controls. Seventeen fragments 433-933 bp long each with 1 to 4 CFTR mutations were prepared using PCR primers containing the mutant sites and flanking paired primers (See FIG. 1, I-VIII). Then 1 to 3 CFTR gene segments were joined (FIG. 1, IX-X) and blunt end cloned as a single fragment into 9 different vectors as illustrated: PCR amplification of the genomic DNA from one of the authors also introduced 3 normal variant sites: 622-194G->A, 1525-61A-G, and 405+46G->T (CFTR Mutation Database). These 9 clones were prepared independently so the desired combinations could be grown and the mutation-carrying fragments mixed according to the selected commercial platform requirements to which the control is applied.
3. Intron10/Exon11 Gene region with the sites of 5 synthesized mutations illustrated. Because the G551D and R553X nucleotide substitution sites are only separated by four basepairs, these mutations were added to different cloned fragments to avoid interference when tested in the multiple formats for which the synthesized mutations were intended.
4. Multiple Intronic Basepair Substitutions Downstream of A455E and N1303K in One Clone. The normal CFTR sequence is shown in the bottom (Subject) row and the initially analyzed cloned sequence in the top (Query) row. Top: The seven nucleotide substitutions found when sequencing the first exon9/intron 9 downstream clone were the same as the substitutions found in the chromosome 20 CFTR pseudogene. All the nucleotide substitutions between the pseudogene and the active CFTR gene are indicated in bold above the top (Query) line. Bottom: The eleven nucleotide substitutions found when sequencing the first exon 21/intron 21 clone. These eleven substitutions occurred in a 130 bp repeated sequence found 162 basepairs downstream from this site.
5. Innogenetics Nitrocellulose Filter Strip Results: Each of the clones was analyzed independently for the synthesized and cloned homozygous mutations. In every case the homozygous mutation was unambiguously distinguished (f1-f8). Note the positive test signal for the homozygous 1717-1 mutation was more intense than the three homozygous signals for G542X, G551D, and R560T which are carried by the same PCR amplified fragment (f3,left). We interpret this signal intensity difference to be a characteristic of the Innogenetics multiplex PCR amplification, but the results are readily interpretable. When these 9 clones are mixed together and tested, all the tested homozygous mutant sites gave homozygous results (mix) except the G551D locus which is mutant in the top cloned fragment (FIG. 1) and normal in the third cloned fragment (FIG. 1) with the R553X mutant site. Therefore the primer to the normal G551D site binds sufficiently to give a positive signal in the presence of normal G551 sequence even though the R553X mutant sequence is upstream. However, the R553X primer only detects mutant sequence in this mixture and not the normal R553 signal on fragment 1 (FIG. 1) because the G551D signal interferes with the binding of the normal R553 reporter molecule in the test kit. In the end, two different Innogenetics test strips define the specificity of all the homozygous controls at every site except the G551D site which gives a heterozygous signal. In order to have all multiplex test sites tested for the specificity of a homozygous mutant result, fragments 1 and 3 (FIG. 1) must be prepared and tested in separate mixes of the other controls.
6. Entire Mixture of Homozygous Control Clones Tested on TmBiosciences Platform. Each homozygous control gives a clearly abnormal result including the 5T locus which has 98% of the fluorescence among the 5T, 7T, and 9T beads carrying specific oligonucleotides for each of these loci (Left Panel, top 3 locations). At least 85% of the signal bound to the homozygous location at all but one tested homozygous control site. Compare this to the 8 normal site results on these fragments where the TmBiosciences test assays for additional mutations (Left) and the normal DNA which gives only normal results at each tested site including the homozygous 7T mutation.

Claims

1. A method of optimizing quality control in a genetic test assay, the method comprising the steps of:

testing for the presence of a normal gene nucleotide sequence portion at a pre-selected gene locus;

testing for the presence of at least a first mutant gene nucleotide sequence portion at the pre-selected gene locus; and

testing for interference by at least a first homologous nucleotide sequence portion.

2. The method of claim 1, wherein the genetic test assay is a hybridization based assay.

3. The method of claim 1, wherein the step of testing for interference by homologous nucleotide sequence portion, involves the step of providing a homologous nucleotide sequence control.

4. The method of claim 3, wherein the homologous nucleotide sequence control includes at least a first homologous nucleotide sequence portion which is homologous to the normal gene nucleotide sequence of the pre-selected gene locus, and is devoid of a hybridizing nucleotide sequence portion of the pre-selected gene locus, wherein the hybridizing nucleotide sequence portion is sufficiently large to prevent detection of the pre-selected gene locus by the assay.

5. The method of claim 4, wherein the hybridizing nucleotide sequence portion is the normal gene nucleotide sequence portion.

6. The method of claim 4, wherein the hybridizing nucleotide sequence portion is substantially the entire gene nucleotide sequence of the pre-selected gene locus.

7. A control comprising:

At least a first nucleotide sequence, wherein the at least a first nucleotide sequence includes at least a first homologous nucleotide sequence portion and wherein the at least a first nucleotide sequence lacks a sufficiently large segment of the gene nucleotide sequence at an at least a first pre-selected gene locus to preclude detection of the gene nucleotide sequence at the at least a first pre-selected gene locus by an assay, and

Adapted for use in the assay, wherein the assay is for the detection of mutations in the gene nucleotide sequence at the at least a first pre-selected gene locus.

8. The control of claim 7, wherein the at least a first nucleotide sequence is total genomic DNA having the sufficiently large segment of the gene nucleotide sequence at the at least a first pre-selected gene locus removed.

9. The control of claim 8 further comprising:

at least a second nucleotide sequence containing a sufficiently large segment of an at least a first pre-selected mutant gene nucleotide sequence so as to be detectable by the assay.

10. The control of claim 9 wherein the at least a second nucleotide sequence contains sufficiently large segments of at least second and third pre-selected mutant gene nucleotide sequences so as to be detectable by the assay.

11. The control of claim 10 wherein the number of copies of the at least a first nucleotide sequence is approximately equal to the number of copies of the at least a second nucleotide sequence.

12. A control comprising:

a first nucleotide sequence portion, wherein the first nucleotide sequence portion includes at least a first homologous nucleotide sequence portion having a sufficient length to adequately imitate corresponding normal or mutant nucleotide sequence portions found at a pre-selected gene locus being tested in an assay and containing at least a first distinct nucleotide species, wherein the at least a first distinct nucleotide species is not found in either the normal or mutant nucleotide sequence portions and wherein the at least a first distinct nucleotide species is suitably distinct as to provide a means for confirmation that the first homologous nucleotide sequence portion is not being detected in the assay by a normal or a mutant sequence primer used in the assay; and

a second nucleotide sequence portion, wherein the second nucleotide sequence portion includes a sufficiently large segment of a first mutant gene nucleotide sequence portion found at the pre-selected gene locus so as to be detectable by the assay.

13. The control of claim 12, further comprising:

at least a second homologous nucleotide sequence portion substantially adjacent the first nucleotide sequence portion, wherein the at least a second homologous nucleotide sequence portion has a sufficient length to adequately imitate corresponding normal or mutant nucleotide sequence portions of the pre-selected gene locus being tested in the assay and containing at least a first distinct nucleotide species, wherein the at least a first distinct nucleotide species is not found in either the normal or mutant nucleotide sequence portions and wherein the at least a first distinct nucleotide species is suitably distinct as to provide a means for confirmation that the at least a second homologous nucleotide sequence portion is not being detected in the assay by a normal or a mutant sequence primer used in the assay

14. The control of claim 13, wherein the second nucleotide sequence portion further comprises:

A sufficiently large segment of an at least a second mutant gene nucleotide sequence portion found at the pre-selected gene locus so as to be detectable by the assay