Isolation of anonymous DNA sequences from within a submicroscopic X chromosomal deletion in a patient with choroideremia, deafness, and mental retardation.

205

Bread wheat (Triticum aestivum L. em Thell) is well suited for cytogenetic analysis because the genome, buffered by polyploidy, can tolerate structurally and numerically engineered chromosomes for analysis over infinite generations. This feature of polyploidy can be used in developing a high-resolution, cytogenetically based physical map of the wheat genome. We show that numerous deletions, observed in the progeny of a monosomic addition of a chromosome from Trtcum cylinduicum in wheat, result from single breakpoints and a concomitant loss ofdistal fragments. Breakages occurred in euchromatic and heterochromatic regions. Forty-one deletions for chromosomes 7A, 7B, and 7D, and a set of genetically mapped DNA probes, were used to construct physical maps. Recombination was low in proximal chromosomal regions and very high toward the distal ends. Deletion mapping was more efficient than genetic mapping in resolving the order of proximal loci. Despite variation in size and arm ratio, relative gene position was largely conserved among chromosomes 7A, 7B, and 7D and a consensus group 7 physical map was constructed. Several molecularly tagged chromosome regions (MTCRs) of approximately one to a few million base pafrs were identified that may be resolved by long-range mapping of DNA frag-

Section: Resultsmentioning

confidence: 99%

Toward a cytogenetically based physical map of the wheat genome.

Werner

Endo

Gill

1992

205

“…Several laboratories have recently published methods for enrichment of deleted DNA sequences from genomic DNA (14)(15)(16)(17)(18). The genomic subtraction procedure described in our paper makes it possible to achieve greater enrichments than was possible using the earlier methods.…”

Section: Discussionmentioning

confidence: 99%

Genomic subtraction for cloning DNA corresponding to deletion mutations.

Straus

Ausubel

1990

189

We have developed a technique, called genomic subtraction, for isolating the DNA that is absent in deletion mutants. The method removes from wild-type DNA the sequences that are present in both the wild-type and the deletion mutant genomes. The DNA that corresponds to the deleted region remains. Enrichment for the deleted sequences is achieved by allowing a mixture of denatured wild-type and biotinylated mutant DNA to reassociate. After reassociation, the biotinylated sequences are removed by binding to avidincoated beads. This subtraction process is then repeated several times. In each cycle we hybridize the unbound wild-type DNA from the previous round with fresh biotinylated deletion mutant DNA. The unbound DNA from the final cycle is ligated to adaptors and amplified by using one strand of the adaptor as a primer in the polymerase chain reaction. The amplified sequences can then be used to probe a genomic library. We applied genomic subtraction to a yeast strain that has a 5-kilobase deletion, corresponding to 1/4000th of the genome.In the experiment reported here, three rounds of subtraction were sufficient to accurately identify genomic clones containing sequences that are missing in the deletion mutant. We discuss the limitations and some potential applications of the method. We describe a technique called genomic subtraction that provides a useful addition to the approaches mentioned above. The procedure identifies clones that contain sequences that are missing in a deletion mutant. It is not labor intensive and should be widely applicable, although it requires the preexistence ofa homozygous deletion mutant that is viable. This paper demonstrates that genomic subtraction can be used to efficiently isolate the DNA that is absent in a yeast deletion mutant. For the preparation of yeast DNA, pellets from 4 liters of saturated culture were resuspended in 240 ml of sorbitol at 167 mg/ml/0.2% (vol/vol) 2-mercaptoethanol/zymolase at 0.1 mg/ml (105 units/mg; Seikagaku America, Saint Petersburg, FL)/100 mM EDTA. After incubation for 1 hr at 370C the cells were spun for 20 min at 40C in a Beckman JS4.2 rotor at 4200 rpm. The pellets were resupended in 60 ml of 50 mM glucose/25 mM Tris'HCl, pH 8/10 mM EDTA. To the resulting 100-ml suspension we added 25 ml of 3.5 M NaCl/ 0.1 M EDTA/0.5 M Tris HCl, pH 8. DNA was prepared from this suspension using a CTAB extraction procedure (1). The pellet from the final EtOH precipitation step was resuspended in 10 ml of 10 mM Tris'HCl, pH 8/1 mM EDTA (TE). MATERIALS AND METHODSRNase A (Sigma) (2) was added at 100 ,ug/ml and the solution was incubated at 370C for 1 hr. The solution was brought to 0.3 M NaOAc and extracted twice with phenol/chloroform (1:1). After adding 2 volumes ofEtOH, DNA was spooled out of the solution, resuspended in 5 ml of TE, and then brought to 0.3 M NaOAc. DNA was spooled out of EtOH solution twice more, washed in 100% EtOH, and resuspended in TE.

“…Kunkel et al (12) used a variation of this method to clone fragments of the Duchenne muscular dystrophy (DMD) locus, which becomes deleted in some afflicted individuals. Clones from other X-chromosome linked deletions have also been obtained by this method (13). We describe here another method for genomic difference cloning that is at least as powerful.…”

mentioning

confidence: 99%

“…The yield of this method is poor, since it depends upon the slow reannealing of dilute tester to itself, and the theoretical enrichment cannot exceed the mass ratio of driver to tester. Yields can be improved through the use of accelerated annealing conditions, such as the phenol emulsion reannealing technique (12)(13)(14).…”

Section: Introductionmentioning

confidence: 99%

A method for difference cloning: gene amplification following subtractive hybridization.

Wieland

Bolger

Asouline

et al. 1990

We describe a procedure for genomic difference cloning, a method for isolating sequences present in one genomic DNA population ("tester") that is absent in another ("driver"). By subtractive hybridization, a large excess of driver is used to remove sequences common to a biotinylated tester, enriching the "target" sequences that are unique to the tester. After repeated subtractive hybridization cycles, tester is separated from driver by avidin/biotin affinity chromatography, and single-stranded target is amplified by the polymerase chain reaction, rendering it double-stranded and clonable. We model two situations: the gain of sequences that result from infection with a pathogen and the loss of sequences that result from a large hemizygous deletion. We obtain 100-to 700-fold enrichment of target sequences.One common and fundamental problem of molecular biology confronts us when two similar genomes differ and we desire to understand the difference. One simple form ofthis problem can occur when a genome becomes deleted for sequences present in another due to germ-line mutation, as can happen in genetic disease (1-3), or due to somatic mutation, as can happen during the development of cancer (4-6). Differences can also be acquired by infection with a DNA-based pathogen. Methods for identifying and isolating sequences present in one DNA population that are absent or reduced in another are called "difference cloning." Methods for difference cloning in cDNA populations have been widely described (7)(8)(9)(10) We make frequent use of the following nomenclature. Two DNA populations that differ are referred to as "tester" and "driver." Tester contains "target" sequences that are not present in driver. In the procedure of Lamar and Palmer (11), target sequences were enriched relative to the remainder of tester in the following way. Tester DNA was prepared by cleavage with Sau3A and mixed with an excess amount of randomly sheared driver DNA. DNAs were melted and reannealed to high Cot values. Double-stranded DNA (ds-DNA) was then cloned into the BamHI site of a cloning vector. In principle, only tester DNA annealed to itself would be clonable into a BamHI site. Neither driver annealed to itself, nor tester to driver, would be clonable. Thus cloned material would be enriched in target since it can form duplex only with itself. The yield of this method is poor, since it depends upon the slow reannealing of dilute tester to itself, and the theoretical enrichment cannot exceed the mass ratio of driver to tester. Yields can be improved through the use of accelerated annealing conditions, such as the phenol emulsion reannealing technique (12)(13)(14).Our procedure utilizes a different form of subtractive hybridization (see Fig. 1). Tester DNA is specially prepared (cleaved, biotinylated, and ligated to "template" oligonucleotides). Prepared tester is then mixed with an excess of randomly sheared driver, melted, and annealed. After annealing proceeds to 90% completion for driver, the remaining single-stranded DNAs (ssDNAs) ...