If the Dna Sequence Ttcacg Is Transcribed to Aaggc, What Type of Mutation Has Occurred?
Nucleic Acids Res. 2002 Jun xv; 30(12): e52.
Improved detection of pocket-size deletions in complex pools of DNA
Anil D'Souza
Biotechnology Laboratory and Section of Zoology, University of British Columbia, Vancouver, BC V6T 1Z4, Canada and oneDepartment of Molecular and Cell Biological science, Oklahoma Medical Research Foundation, 825 Northward.Due east. 13th Street, Oklahoma City, OK 73104, U.s.
Gary Moulder
Biotechnology Laboratory and Department of Zoology, University of British Columbia, Vancouver, BC V6T 1Z4, Canada and iSection of Molecular and Jail cell Biology, Oklahoma Medical Research Foundation, 825 Northward.E. 13th Street, Oklahoma City, OK 73104, USA
Robert Barstead
Biotechnology Laboratory and Department of Zoology, University of British Columbia, Vancouver, BC V6T 1Z4, Canada and aneDepartment of Molecular and Cell Biology, Oklahoma Medical Research Foundation, 825 Due north.E. 13th Street, Oklahoma City, OK 73104, USA
Received 2001 Dec 14; Revised 2002 Apr xvi; Accepted 2002 Apr sixteen.
Abstract
Most 40% of the genes in the nematode Caenorhabditis elegans have homologs in humans. Based on the history of this model system, information technology is clear that the awarding of genetic methods to the written report of this set of genes would provide important clues to their function in humans. To facilitate such genetic studies, we are engaged in a project to derive deletion alleles in every gene in this set. Our standard methods make use of nested PCR to hunt for animals in mutagenized populations that carry deletions at a given locus. The deletion bearing animals exist initially in mixed populations where the majority of the animals are wild type at the target. Therefore, the product of the PCR fragment representing the deletion allele competes with the product of the wild type fragment. The size of the deletion fragment relative to wild type determines whether information technology can compete to a level where it can be detected above the background. Using our standard conditions, we have plant that when the deletion is <600 bp, the deletion fragment does not compete effectively with the production of the wild type fragment in PCR. Therefore, although our standard methods work well to detect mutants with deletions >600 bp, they exercise not work well to observe mutants with smaller deletions. Here we report a new strategy to detect small deletion alleles in complex DNA pools. Our new strategy is a modification of our standard PCR based screens. In the first circular of the nested PCR, we include a 3rd PCR primer between the two external primers. The presence of this tertiary primer leads to the production of iii fragments from wild type Dna. We configure the system then that two of these three fragments cannot serve as a template in the 2nd round of the nested PCR. The addition of this third primer, therefore, handicaps the amplification from wild type template. On the other hand, the distension of mutant fragments where the binding site for the third primer is deleted is unabated. Overall, we run into at least a 500-fold increase in the sensitivity for small deletion fragments using our new method. Using this new method, nosotros written report the recovery of new deletion alleles within 12 C.elegans genes.
INTRODUCTION
Until recently, most genes in the nematode Caenorhabditis elegans were known through their mutant phenotypes. Now, withal, more C.elegans genes are known through sequencing than through genetics (1); of the approximately xix 000 genes in C.elegans, only well-nigh 1300 accept mutant alleles. Every bit genetics is ane of the about important tools in the arsenal of C.elegans biologists, we and others have devised methods to derive mutations in C.elegans genes known merely through their sequence (2–6). Our present methods are conceptually simple. We treat worms at the L4 larval stage with a mutagen. The progeny of the mutagenized worms are subdivided into populations that are allowed to reproduce. Nosotros and so extract the Deoxyribonucleic acid from ∼30% of each population. The extracted Dna samples are pooled and subjected to PCR with nested primer sets (Fig. 1). Candidate populations are identified by the presence of a PCR product that is smaller than the size predicted by the genomic Dna sequence. Each candidate population is subdivided and subjected to like growth and PCR analysis. This process of sib selection continues until we recover a unmarried individual with the deletion. Using this protocol, typically we can recover such an individual in iii steps of growth and sib selection.
Protocol schematic. Our electric current protocols use nested PCR to place smaller than normal PCR fragments derived from a mutagenized population of worms. Typically, nosotros use two external (A and C) and two internal PCR primers (D and Eastward). As shown, the poison primer method adds a primer (B) in the first circular of the nested PCR.
In principle, our ability to notice a deletion should be a function of the resolution of the gels used for electrophoresis. In practice, however, gel resolution does non determine the size of the deletions that we identify; typically, we exercise not detect deletions that are less than ∼600 bp. This limitation is a consequence of our endeavor to reduce, as far every bit possible, the cost and time required to identify a candidate deletion. To accommodate the relatively depression frequency at which deletions are induced past chemical mutagens (7), we puddle the DNA template samples to minimize the numbers of PCRs that are needed to detect a deletion. Using our present DNA pooling strategy, for every copy of the deletion Dna we have approximately 12 000 copies of wild type DNA. This places the mutant template at a substantial disadvantage in PCR. Deletion fragments that are close to the size of wild type Dna apparently are non able to overcome this initial disadvantage.
However, recent work has shown that a meaning proportion of deletions induced by trimethylpsoralen (TMP) treatment followed by UV irradiation are <600 bp, falling in the range of 50–600 bp (E.Gilchrist, M.Edgley, M.Mullen, B.Shen, T.Rogalski, T.Szczygielski and D.Moerman, manuscript in preparation) (8). Based on these data, it is likely that we do not detect a pregnant number of TMP-induced deletion mutants using our standard screening strategy. The challenge then became to develop methods that would let united states to discover small deletions. This paper reports such a method.
MATERIALS AND METHODS
Reconstruction experiments
Our initial tests were done with a known 133 bp deletion in the C.elegans factor dim-1(gk54) (GenBank locus {"blazon":"entrez-nucleotide","attrs":{"text":"U39667","term_id":"1049412","term_text":"U39667"}}U39667). We used the following oligonucleotide primers in nested PCR every bit shown in Figure i: first round, 5′-CTCAGTCGATCACAGTACA-iii′, 5′-TCCACCAACAAGCTTTTGCC-3′; second round, 5′-ACACTTCCCACAACAACCAG-3′, 5′-CGGTAAGCTTCAGGTTGAAG-3′; internal poison, (A) five′-CGAACAAGGGAAGCGACAGC-3′, (B) five′-GTTGGCACTGAAGCGTCCAG-3′.
Each primer was used at a final concentration of i µM. Nosotros did PCR using template Deoxyribonucleic acid purified from wild blazon or dim-i mutant strains. Each of the necessary deoxynucleotide triphosphates was at a concentration of 250 µM. The PCR cycling parameters were as follows: 94°C, 30 south; sixty°C, 30 south; 72°C, 90 s. We did 35 total cycles. PCR fragments were analyzed on ane% agarose gels.
Generation and screening of mutant libraries
To induce deletion mutations, immature developed hermaphrodites were treated with trimethylpsoralen and UV light as described (seven) (East.Gilchrist, M.Edgley, Yard.Mullen, B.Shen, T.Rogalski, T.Szczygielski and D.Moerman, manuscript in preparation). Fone progeny of mutagenized animals were cultured in 1152 groups of 50 worms each. Subsequently one generation, Dna was prepared from each population by proteinase 1000 lysis. PCR was used to identify populations with animals carrying deletions using the cycling parameters described below. Populations conveying a deletion were repeatedly subdivided until homozygotes carrying the deletion were obtained. Deletion endpoints were determined by sequencing PCR products that spanned the deleted region.
PCR conditions
External-circular screening PCRs were x µl and contained 2 µl library Deoxyribonucleic acid, iv pM each external primer and poison primer, 200 µM each dNTP, i× standard PCR buffer containing one.v mM MgClii and 0.2 U Taq polymerase (Roche). Internal-round PCRs were identical in all respects except for input DNA (0.2 µl from external round) and primers (four pM each internal primer). Replication of the external round into the internal round was done with a Robbins Hydra 96 pipetting station. Thermal cycling was done on MJ Enquiry PTC-200 Deoxyribonucleic acid Engines or Biometra Uno II thermal cyclers using the following weather: 94°C/30s, 35 cycles of 94°C/30 s – 61°C/30 s (external round) or 55°C/xxx s (internal round) – 72°C/60 s, followed by cooling to four°C. Loading buffer (10 µl; fifteen% Ficoll with bromophenol blueish and xylene cyanol) was added to each reaction, and 1 µl of this mix was loaded into each lane of 2% agarose gels. Gels were stained for 30 min with fresh 1:ten 000 SYBR® Green (BMA) and imaged with a Molecular Dynamics Fluorimager. The molecular weight marker is BioRad 100 bp PCR Molecular Ruler (Cat. 170-8206). The wild type amplification product is 1151 bp and the deletion product is 810 bp.
RESULTS
Our initial experiments were done with a known pocket-sized deletion in the C.elegans gene dim-i (T.Rogalski and D.Moerman, unpublished information). We showtime tested dissimilar pooling strategies, hoping to reduce the competition between the deletion and wild type fragments. We plant that a 133 bp deletion could exist detected merely if the initial ratio of wild type to deletion template did not exceed 10:1 (Fig. ii). Together with the forrad frequency for PCR detectable deletions generated by TMP (probably <ane × 10–5 for the average gene) this indicates that an unreasonably large number of PCRs would be required to discover a minor deletion. It was not viable, therefore, simply to reduce the pooling depth to improve our ability to discover small deletions.
Poison primers improve sensitivity for small deletions. Nosotros tested the poison primer method on a known deletion mutant. Nosotros did nested PCR using ii different internal poison primers, designated competing oligos A and B that were known to fall within the deleted sequence. Different amounts of wild type and deletion mutant DNAs were mixed at the indicated ratios. With no poison primer, wild type gives a 701 bp PCR fragment. The deletion mutant gives a 568 bp fragment. Without the poisonous substance primer, we lose the ability to detect the deletion at wild blazon:mutant ratios between x:one and 100:1. With the poison primer, we tin can detect the deletion fragment at ratios of 5000:1.
Reconstruction experiments with known deletion alleles
To better the sensitivity of the PCR assay for deletion fragments that are shut to the wild blazon size, nosotros developed the strategy shown in Figure 1. This strategy is a modification of our original protocol, in which we used a two-stride nested PCR series to observe deletions betwixt the primer sets. In the modified version, a third functional PCR primer that falls between the two external primers is included in the first circular of nested PCR. Amplification from the wild blazon template leads to the product of 2 fragments, one full length and the other relatively curt. In do, the shorter fragment is produced much more than efficiently than the longer. Amplification from a mutant template, in which the site for the third internal primer is deleted, leads to the production of a unmarried mutant fragment from the external primers. In the 2nd circular of PCR, we apply two primers placed just inside the external starting time circular primers. The shorter wild blazon band from the first round cannot serve as a template for the second round PCR because it does not include one of the 2d round primer bounden sites. The longer wild blazon fragment can serve as a template, merely because its product was limited by competition in the first round, its production in the second round is limited correspondingly. The internal functional primer is called a 'poison' because it interferes with product of the full-length wild type fragment. The effectively lower level of wild type product gives the deletion fragment an reward. Our information show that nosotros can detect a 133 bp deletion at wild type to mutant ratios of 5000:1 (Fig. 2).
Screens for new alleles
We tested our new strategy in screens for new deletion mutants. We cultured the Fone progeny of mutagenized parents in sets of ten or fifty worms. We harvested a portion of each F2 population and prepared Dna for PCR. Although, as described above, we were able to notice a small-scale 133 bp deletion in pools where the ratio of wild type to mutant DNA reached 5000:ane, we did non exceed a ratio of 1200:1 in screens for new unknown deletions. Figure three shows an example of a 341 bp deletion identified using the poison primer method and a pooling ratio of 240:1. The detection of this deletion required the presence of the poison primer, as the deletion ring was clearly visible when information technology was in the reaction mix but was non detected otherwise (Fig. 3). This instance clearly illustrates the enhanced detection that can exist obtained using this strategy.
Poison primer strategy works to place new deletion alleles. Gel epitome of nested PCR distension products illustrating the consequence of a poison primer in the external distension circular on deletion detection. Twelve samples of pooled library Deoxyribonucleic acid, one of which contained a 341-bp deletion in the gene H32C10.iii at a complexity of one deletion chromosome in 240, were subjected in duplicate to PCR distension with and without a poisonous substance primer that lies inside the deleted region. Starting after the marker lane (Thousand), the two leftmost lanes contain products of reactions with the poisonous substance primer, and the two rightmost lanes incorporate products of reactions without the poison. The wild type and deletion fragment positions are equally indicated.
Nosotros identified 12 deletion strains from a UV/TMP mutagenized library of mutants using the toxicant primer method. The deletions ranged from 78 to 850 bp. Examples of these are shown in Effigy ivA. Figure fourB shows the position of a minor deletion that we recovered in a factor that resides entirely inside the intron of some other gene. Dna sequencing showed that 11 of the 12 deletions embrace the poison primer binding sites. The toxicant primers were required to detect 10 of these 11 deletions in the initial mutant library screens (information not shown). The infrequent case was a deletion of 850 bp in a 1335-bp wild type interval.
Detection of deletions with the poison primer technique. The cistron structure for each of the genes listed was predicted from genomic Deoxyribonucleic acid sequence. Greyed boxes and sparse lines stand for exons and introns, respectively. The positions of the deletions and their sizes are shown beneath each factor. Arrows represent the poisonous substance primers designed to interfere with the amplification of a full-length wild blazon PCR product. Detail regions of the gene can be targeted based on the placement of such primers. (A) Representative deletion mutations. Deletion breakpoints were adamant past DNA sequencing of the internal PCR products amplified from DNA isolated from single worms. The deletions shown are 342 NT, 769 NT, 483 NT and 369 NT in the coding regions of H32C10.3, C01G8.2, T19E10.1a and Y56A3A.20, respectively. Note that the deleted regions contain all or function of the poison primer-binding site. (B) Precise excision of a nested cistron. R13H4.ane is a large cistron whose coding sequence is on the minus strand of clone R13H4 of chromosome II of C.elegans. The factor R13H4.2, one of ii genes nested inside introns of the larger gene on the opposite strand, was targeted with two oppositely oriented toxicant primers. The deletion mutation detected with these primers excised the entire 3′ stop of R13H4.ii, including the target exon, just did not extend exterior of the intron of the larger cistron, leaving its coding sequence intact.
Word
Genome sequence data provide a platform for standard mutational genetics, a powerful strategy to examine gene function. To exploit genetic methods, even so, i must place mutations at target loci. Using our original protocols, we could detect deletions >600 bp in an interval of 3000 bp. Using the poison primer protocol, we accept detected deletions equally modest every bit 78 bp. We as well tested whether we could improve the method through the simultaneous use of two closely spaced poison primers annealing to opposite strands of the target. Although our data set is small, ii poisons appeared to be somewhat better at reducing the level of the competing wild type PCR product (data not shown).
Although the poison primer method allows for the detection of small deletion alleles in complex DNA pools, it changes the target for deletion relative to our typical strategy. With the poisonous substance primer method the deletion must eliminate the internal poison primer site. In a 3000 bp interval, therefore, with a poison annealing to a unmarried site i can detect just ∼seven% of the total number of 100 bp deletions, 14% of the 200 bp deletions, etc. The poison primer method, nonetheless, gives greater command over the position of the deletion within the target, thereby assuasive for the recovery of more precise 'designer' deletions. Cases where this method might be useful include the deletion of a single exon in alternatively spliced genes, the deletion of parts of promoters, or the deletion of genes that fall within introns of other genes. The data in Figure 4B validate the poison primer method for such uses.
Although the verbal endpoints of the deletions are not discipline to our control, nosotros by and large target the 5′ end of the coding sequence to heighten the odds that the recovered deletion alleles will eliminate all functions of the gene. We accept developed a public interactive web site that can be used to place primer sequences that meet our pattern criteria (http://ko. cigenomics.bc.ca/oligos.shtml).
We have used the nematode C.elegans to develop a new method to detect deletion mutations in known genes. Our new method enhances the contrary genetic tools bachelor for C.elegans and other genetic model systems and, further, adds to the growing list of genetic tools adult to screen for mutations in humans in advisable clinical or research populations.
ACKNOWLEDGEMENTS
Nosotros thank Marco Marra and Steven Jones of the Genome Sequence Heart in Vancouver BC for their advice and enthusiasm for this projection, and for the use of their fluorimager to scan gels. This work was supported by a National Institutes of Health grant R01 HG01843 to R.B., and a grant from the Canadian Institute for Wellness Enquiry to D.Thousand.M. Boosted support was provided by the Biotechnology Laboratory and U.B.C. to the C.elegans Reverse Genetics Core Facility.
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