Chromosome-band-specific painting: chromosome in situ suppression hybridization using PCR products from a microdissected chromosome band as a probe pool

Summary We describe a chromosome-band-specific painting method that involves (1) microdissection of the chromosome, chromosomal region or band, (2) amplification of a variety of chromosome/region/band-specific DNA fragments with the polymerase chain reaction (PCR), and (3) chromosome in situ suppression hybridization (CISS) with the direct use of the PCR products as a probe pool. With this method, it was possible 1) to paint an entire X or Y chromosome, a distal one-fourth of 2q, and only a band at 8q24.1, 2) to identify the origin of a minute marker chromosome in a mentally retarded patient, 3) to detect an X;Y translocation in another patient, and 4) to identify one human chromosome 2 in a human-mouse hybrid cell line. This method allows us to identify not only structural chromosome abnormalities at the band level, but also the origin of cytogenetically unidentifiable marker chromosomes. It will also be useful in studies of evolutionary cytogenetics.     

PCR amplification of microdissected wheat chromosome arms in a simple 'single tube' reaction

A novel method for the adaptor-mediated PCR amplification of microdissected chromosome arms is described. This simple and versatile protocol eliminates the need for enzymatic micromanipulation in nanoliter volumes and permits the efficient amplification of as little as two wheat chromosome arms in a 'single tube' reaction.     

Laser microdissection and single unique primer PCR allow generation of regional chromosome DNA clones from a single human chromosome.

We have developed an argon laser chromosome microdissection technique in conjunction with a polymerase chain reaction (PCR) approach to directly amplify microdissected chromosomes. The single 22-mer primer used in PCR, although unique in sequence (5'-TAGATCTGA-TATCTGAATTCCC-3'), randomly primed and amplified any target DNA. These methods were applied to the distal half of the short arm of human chromosome 4 containing the Huntington disease (HD) locus. Forty-four percent of representative clones from this library identify single-copy DNA sequences. This calculation suggests that the resulting chromosome-specific DNA library contains approximately 600 nonoverlapping sequences with an average size 350 bp at an average spacing of 30 kbp along chromosome 4. This microdissection and PCR cloning procedure is a simple and general approach for constructing a chromosome region-specific DNA library from a single metaphase spread.     

Isolation of 24 novel cDNA fragments from microdis—sected human chromosome band

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Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer.

A version of the polymerase chain reaction (PCR), termed degenerate oligonucleotide-primed PCR (DOP-PCR), which employs oligonucleotides of partially degenerate sequence, has been developed for genome mapping studies. This degeneracy, together with a PCR protocol utilizing a low initial annealing temperature, ensures priming from multiple (e.g., approximately 10(6) in human) evenly dispersed sites within a given genome. Furthermore, as efficient amplification is achieved from the genomes of all species tested using the same primer, the method appears to be species-independent. Thus, for the general amplification of target DNA, DOP-PCR has advantages over interspersed repetitive sequence PCR (IRS-PCR), which relies on the appropriate positioning of species-specific repeat elements. In conjunction with chromosome flow sorting, DOP-PCR has been applied to the characterization of abnormal chromosomes and also to the cloning of new markers for specific chromosome regions. DOP-PCR therefore represents a rapid, efficient, and species-independent technique for general DNA amplification.     

Cloning regions of the Drosophila genome by microdissection of polytene chromosome DNA and PCR with nonspecific primer.

A simple and rapid procedure to isolate clones carrying sequences from a specific region of the polytene chromosome of Drosophila is demonstrated. The procedure involves microdissection of the region of interest, amplification of the DNA by PCR using a primer designed to prime the synthesis nonspecifically, labeling of the amplified DNA using the random primer method, and screening of a standard library with the probe to identify and isolate clones carrying sequences homologous to the dissected region. This procedure has the potential to replace the difficult procedure of microcloning, as well as facilitate chromosome walking.     

Genomic DNA amplification from a single bacterium

Genomic DNA was amplified about 5 billion-fold from single, flow-sorted bacterial cells by the multiple displacement amplification (MDA) reaction, using phi 29 DNA polymerase. A 662-bp segment of the 16S rRNA gene could be accurately sequenced from the amplified DNA. MDA methods enable new strategies for studying non-culturable microorganisms.     

Rapid and economic DNA extraction from a single salmon egg for real-time PCR amplification

Salmon eggs are common in Japanese sushi and other seafood products; however, certain fish eggs are used as counterfeit salmon eggs which are found in foods and processed products. This study develops a simple, rapid, and cost-effective method for DNA extraction, filtration (FT) and dilution (DL) protocols from a single salmon egg with good DNA quality for real-time PCR amplification. The DNA amount, DNA quality, and real-time PCR performance for different dilutions and different lengths of PCR amplicons were evaluated and compared with the common Qiagen tissue kit (QTK) and Chelex-100-based (CX) protocols. The extracted DNA from a single salmon egg using the FT or DL protocol can be applied in phylogenic research, food authentication and post-marketing monitoring of genetically modified (GM) food products.     

PCR in situ followed by microdissection allows whole chromosome painting probes to be made from single microdissected chromosomes

Whole-chromosome painting probes (WCPs) and chromosome-arm painting probes (CAPs) are an integral part of the cytogenetic analysis of chromosome abnormalities. While these are routinely made by chromosome microdissection, multiple copies of the dissected region have been necessary to achieve a library sufficiently complex to provide adequate painting. Performing multiple dissections of chromosomes or chromosome regions is time consuming and occasionally impossible, such as when working with species whose banded karyotype is not well defined. We have developed a method whereby chromosome paints can be reliably generated by dissecting single chromosomes. The technique consists of performing degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) in situ on the chromosomes, prior to dissection. Enough amplification occurs to enable a single dissected chromosome to be used to create a painting probe sufficiently complex for use in fluorescence in situ hybridization (FISH). The amplification products remain localized on the chromosomes; this allows region-specific chromosome paints to be made. We detail this novel technique and show whole-chromosome, arm-specific, and contiguous region-specific probes for human and rat, each created from single dissected fragments of chromatin. Received: 14 January 1999 / Accepted: 28 January 1999     

QuantiLyse: reliable DNA amplification from single cells

Amplification of DNA sequencesfrom single cells via PCR is increasingly used in basic research and clinical diagnostics but remains technically difficult. We have developed a cell lysis protocol that uses an optimized proteinase K solution, named QuantiLyse and permits reliable amplification from individual cells. This protocol was compared to other published methods by means of real-time PCR with molecular beacons. The results demonstrate that QuantiLyse treatment of single lymphocytes renders gene targets more availablefor amplification than other published proteinase K methods or lysis in water. QuantiLyse and an optimized alkaline lysis were equally effective in terms of target availability, although QuantiLyse offers greaterflexibility, as it does not require neutralization and can comprise a higher percentage of the final PCR volume. Maximum gene target availability is also obtained following QuantiLyse treatment of samples containing up to 10000 cells (the largest number tested). Thus, QuantiLyse maximizes the chances that targeted DNA sequences will be available for amplification during the first cycle of PCR, thereby reducing the variability among replicate reactions as well as the likelihood of amplification failure or allele drop-out. QuantiLyse will be useful in a range of investigations aimed at gene detection in small numbers of cells.     

Preparation of DNA suitable for PCR amplification from fresh or fixed single dinoflagellate cells

A method is described to prepare total DNA from single cells of dinoflagellates, which can be used for PCR amplification. As model organisms, we used a stock strain of Alexandrium catenella and cells of Dinophysis acuminata harvested from the Atlantic Ocean. Fresh grown cells or cells maintained in different preservatives were tested as sources for DNA preparation. The method used to prepare DNA combines physicochemical and enzymatic procedures on cells embedded in agarose plugs or beads. The agarose pieces containing the DNA were used to perform PCR amplification of a fragment of DNA containing a 5.8S rRNA gene and the flanking internal transcribed spacers (ITS1 and ITS2).     

Multiple displacement amplification of the DNA from single flow–sorted plant chromosome

A protocol is described for production of micrograms of DNA from single copies of flow‐sorted plant chromosomes. Of 183 single copies of wheat chromosome 3B, 118 (64%) were successfully amplified. Sequencing DNA amplification products using an Illumina HiSeq 2000 system to 10× coverage and merging sequences from three separate amplifications resulted in 60% coverage of the chromosome 3B reference, entirely covering 30% of its genes. The merged sequences permitted de novo assembly of 19% of chromosome 3B genes, with 10% of genes contained in a single contig, and 39% of genes covered for at least 80% of their length. The chromosome‐derived sequences allowed identification of missing genic sequences in the chromosome 3B reference and short sequences similar to 3B in survey sequences of other wheat chromosomes. These observations indicate that single‐chromosome sequencing is suitable to identify genic sequences on particular chromosomes, to develop chromosome‐specific DNA markers, to verify assignment of DNA sequence contigs to individual pseudomolecules, and to validate whole‐genome assemblies. The protocol expands the potential of chromosome genomics, which may now be applied to any plant species from which chromosome samples suitable for flow cytometry can be prepared, and opens new avenues for studies on chromosome structural heterozygosity and haplotype phasing in plants.     

PCR amplification of chromosome-specific DNA isolated from flow cytometry-sorted chromosomes.

We have established a method for amplifying and obtaining large quantities of chromosome-specific DNA by linker/adaptor ligation and polymerase chain reaction (PCR). Small quantities of DNA isolated from flow cytometry-sorted chromosomes 17 and 21 were digested with MboI, ligated to a linker/adaptor, and then subjected to 35 cycles of PCR. Using this procedure, 20 micrograms of chromosome-specific DNA can be obtained. Southern blot analysis using several DNA probes previously localized to chromosomes 17 and 21 indicated that these gene sequences were present in the amplified chromosome-specific DNA. A small quantity of the chromosome-specific DNA obtained from the first round of PCR amplification was used to amplify DNA for a second, third, and fourth round of PCR (30 cycles), and specific DNA sequences were still detectable. Fluorescence in situ hybridization using these chromosome-specific DNA probes clearly indicated the hybridization signals to the designated chromosomes. We showed that PCR-amplified chromosome 17-specific DNA can be used to detect nonrandom chromosomal translocation of t(15;17) in acute promyelocytic leukemia by fluorescence in situ hybridization.     

Identification of a polytene chromosome band containing a male sex determiner of Chironomus thummi thummi

Hybrid males of Chironomus thummi piger x Ch. th. thummi crosses were backcrossed with females of both parental stocks. Fourth-instar larvae of these backcrosses showed sex specific differences in the pairing behavior of region D₃d-g in chromosome arm F of salivary gland chromosome III. — Analysis of the banding pattern of region D₃d-g after RB and quinacrine staining demonstrated that in piger x thummi hybrid males a single selectively stained band occurs within this region in the heterozygous condition at map position D₃e₁. This band could only be found in the thummi chromosome partner, it is heterochromatic and contains AT-rich DNA. In female hybrid larvae, however, such a selectively stained band is present in neither the thummi nor the piger chromosome region D₃d-g. From these results it is concluded that the selectively stained band D₃e₁ represents the male sex determiner of our Ch. th. thummi stock and that the male is the heterogametic sex.     

Microdissection of and microcloning from the short arm of human chromosome 2.

A bank of cloned DNA sequences from the distal half of the short arm of human chromosome 2 was generated by using microdissection and microcloning techniques. DNA was purified from 106 chromosomal fragments, manually dissected from peripheral lymphocytes in metaphase, and cloned into the EcoRI site of lambda gt10. A total of 257 putative recombinants were recovered, of which 41% were found to contain human inserts. The mean insert size was 380 base pairs (median size, 83 base pairs), and fewer than 10% of the clones contained highly repetitive sequences. All single-copy sequences examined were shown to map to the short arm of chromosome 2 by using hybrid panels. This technique provides a rapid method of isolating probes specific to a human subchromosomal region to generate linked markers to genetic diseases for which the chromosomal location is known.