CHROMOSOME MICRODISSECTION AND MICROCLONING PDF

Jump to navigation Jump to search Chromosome microdissection is a technique that physically removes a large section of DNA from a complete chromosome. The smallest portion of DNA that can be isolated using this method comprises 10 million base pairs - hundreds or thousands of individual genes. Scientists who study chromosomes are known as cytogeneticists. They are able to identify each chromosome based on its unique pattern of dark and light bands.

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China; Tel: ; Fax: ; E-mail: nc. This article has been cited by other articles in PMC. Abstract The technique of chromosome microdissection and microcloning has been developed for more than 20 years.

As a bridge between cytogenetics and molecular genetics, it leads to a number of applications: chromosome painting probe isolation, genetic linkage map and physical map construction, and expressed sequence tags generation. During those 20 years, this technique has not only been benefited from other technological advances but also cross-fertilized with other techniques.

Today, it becomes a practicality with extensive uses. The purpose of this article is to review the development of this technique and its application in the field of genomic research. Moreover, a new method of generating ESTs of specific chromosomes developed by our lab is introduced. By using this method, the technique of chromosome microdissection and microcloning would be more valuable in the advancement of genomic research.

Chromosome Recognition-- the Prerequisite of this Technique Scalenghe et al. They used this technique to generate DNA from individual bands of Drosophila melanogaster polytene chromosomes and obtained 80 clones [ 61 ]. This technique was then applied to mouse to obtain microclones from the proximal half of chromosome 17 containing the t-complex [ 56 ]. Then, it was extended to human chromosomes [ 3 ]. However, at that time, studies were mainly focusing on some chromosomes that are easily identifiable by their configuration, such as the X chromosome of mouse [ 5 ] and chromosome 2 of human [ 3 ].

After the advent of G-banding technique, which makes the identification of human and animal chromosomes easier, and PCR technique, the chromosome microdissection and microcloning technique was extensively used in human and animal genomics research [ 38 , 46 , 47 , 49 , 74 , 75 ] To microdissect chromosomes in plant is more difficult than in human, because chromosome preparation is more difficult in plant.

The chromosome microdissection and mi-crocloning technique was applied to isolate B-chromosome DNA from rye in [ 58 ]. It was the first case that this method was used on plant chromosomes.

However, work has been limited to chromosomes that are easy to identify, such as the satellite chromosomes [ 11 , 33 , 80 - 82 ], the largest or the smallest chromosome, and B chromosome [ 27 ]. Chromosomes in cytogenetic stocks that can be easily distinguished were used for chromosome microdissection, such as telo-chromosomes [ 43 , 44 ], addition lines [ 37 , 65 , 22 , 34 , 35 ], and reconstructed translocation chromosomes.

Benefited from other Techniques Chromosome microdissection and microcloning has been benefited from technological advances and coupling with other techniques, which further improved its application. In the microdissection and microcloning technique, the chromosome was initially dissected with glass microneedles under an inverted microscopy [ 61 ].

Even for an expert, it is difficult to dissect and collect a large numbers of chromosome fragments from the same region.

Monajembashi et al. The equipment they used is mainly composed of argon-ion laser power supply, microcomputer and an invert microscope. Even though the intensity and the position of the laser beam were controlled by a microcomputer, the collection of the target region is still a difficult step for the operators.

In our lab, these two methods were combined together. Firstly, we use the laser beam to dissect the targeted region of chromosomes, and then, the glass microneedle was used to collect the targeted regions [ 72 ]. Flow cytometry FCM has been applied to the vast field of cytogenetics research through adaptation to the observation of isolated chromosomes since [ 50 ].

Flow cytometry, while successful in isolating some of the larger chromosomes was clearly limited in its sensitivity for isolating the smaller ones. In order to isolate one special chromosome, Griffin et al.

From glass microneedle to laser microbeam, and then flow cytometric technology, chromosome microdissection method had undergone changes from manual operation to computer driven manipulation.

As a result, both the rate and precision of chromosome isolation are improved. Moreover, these dissection techniques have complementary advantages. The earliest chromosome microcloning [ 61 ] was a kind of direct cloning of the dissected chromosomal material in a nanoliter microdrop contained in an oil chamber. A very large number of dissected chromosome fragments had to be used and a relatively low cloning efficiency was attained.

However, the introduction of PCR technology to the micro-cloning protocols has brought a substantial improvement in cloning efficiency [ 46 ]. It avoids the complex micromanipulation in a microchamber and can generate much larger fragments—the average length is about , bps [ 28 , 80 ].

Using this method, Chen and Arm-strong constructed a single chromosome less than 0. The DOP-PCR technique, which is rapid, efficient, and species-independent, is designed to amplify target DNAs at frequently occurring priming sites using the primer of partially degenerate sequence, without restrictions imposed by the complexity or the origin of DNA.

The combination of improved micromanipulation methods and PCR technology has enabled scientists to dissect specific chromosomes or chromosomal regions both accurately and frequently, thus, improving the efficiency of this technique.

The isolated DNA can be used for genomic research including 1 genetic linkage map and physical map construction, 2 generation of probes for chromosome painting, and 3 generation of chromosome specific expressed sequence tags libraries. Providing Probes for Genetic Linkage Map and Physical Map Construction Despite the rapid progress in gene mapping in recent years, there are still large areas of the genome for which markers are sparse or which are completely unmapped.

Direct chromosomal microdissection and microcloning is a rapid technique for providing probes for such areas. The clones of specific chromosomes libraries can be used in conjunction with existing markers to construct a fine genetic linkage map and physical map, providing a gateway for understanding of chromosome structure and organization of a specific region of the genome.

Combined with chromosome walking, chromosome region-specific physical map can be constructed by using chromosomes or chromosome fragments library to select cosmid library yeast artificial chromosome YAC and bacterial artificial chromosome BAC. The versatility and resolution of FISH depends critically on the probe set used [ 68 ]. The probes usually generate from the clones of cosmid library, yeast artificial chromosome YAC and bacterial artificial chromosome BAC.

Microdissect DNA clones are also the source of chromosome painting probes, particularly in the research of human genome. The technique that combines chromosome microdissection and chromosome painting is named micro-FISH [ 12 , 49 , 21 ].

As one of the important applications of chromosome microdissection, micro-FISH can be used to identify the reliability of the origin of microdissect DNA [ 65 , 70 , 80 ]. On the other hand, micro-FISH is an important tool for other research, such as chromosome construction aberration, chromosome origin identification and comparative analysis of genomes [ 18 ]. More and more chromosomal sites in the human genome have been identified as primary lesions in specific genetic diseases or cancers, such as the 4p Isolating the sequences from human chromosomal regions associated with specific genetic diseases or cancers to understand disease related genes is the prevalent approach in latest clinical research.

Arens et al. Vermeesch et al. To study the behavior of genes conferring drug resistance of cancer lines, Mahjoubi et al. The isolated sequence provided a resource for future investigations in searching for novel genes contributing to drug resistance [ 48 ]. For comparative genome analysis, Rubes et al. Using microdissected plant chromosome DNA, there are many advantages to paint chromosomes from various plant species with large genomes, such as barley [ 17 , 62 ], wheat [ 70 ], Vi-cia faba [ 76 ], and Secale cereale [ 80 ].

However, no specific paintings of chromosomes were observed. The painting probes were obtained only from B chromosomes [ 25 , 26 ] and Y chromosome [ 64 ], because of the abundance of chromosome specific repetitive sequences.

The ESTs give important information about its coding content and expression patterns in different tissues and organs of organisms at different developmental stages and environments. That is why partial cDNA sequencing to generate ESTs is being used at present for the fast and efficient profiling of gene expression in various tissues, cell types, or developmental stages.

If the ESTs could be isolated from those specific chromosomes and specific chromosome regions directly, it would be easier to study and isolate those genes. However, the dataset is huge and highly redundant, and it is difficult to identify the chromosome specific ESTs from cDNA libraries.

Several methods to isolate chromosome specific and chromosome region specific expressed sequences had been developed, mainly including 1 restriction enzyme mapping of CpG islands [ 4 ]; 2 exon trapping [ 6 , 13 , 41 ]; 3 direct selection; 4 Hybrid selection and 5 Microdissection-mediated cDNA capture.

First two of them, however, are labor-intensive and technically complex. Here we will discuss only the last three methods that are being used frequently in recent years. Direct Selection The generation of a regionally specific genomic library is one approach to obtain regionally defined expressed sequences. Such libraries may be constructed by microdissec-tion of small chromosome fragments [ 46 , 47 , 74 ]. Yokoi et al. Hybrid Selection Su et al.

Chromosome microdissection and hybrid selection was combined together in this technique to generate the chromosome-region-specific cDNAs. And then, the denatured amplified DNA was immobilized on a nylon membrane. Using this method, Su et al. After washing off the unhybridized cDNA, the targeted chromosomes or chromosome fragments are dissected by micromanipulation. Then, the microdissect DNA is amplified using the adaptor primers. A New Method of Ests Selection may be more Efficient -- Hybrid Suppressive Amplification Selection It is a new potential approach developed by our lab to rapidly generate ESTs from a specific chromosome or even a chromosome-specific-region paper in preparation.

We combine chromosome microdissection method and hybrid suppressive amplification HSA technique [ 42 ], to yield the homologous sequences between microdissected DNA and cDNA. The two samples were mixed and annealed together for hours. Finally, a two-step PCR amplification is performed to select the hybridized fraction of the samples: Only the hybridized double strand sequences, which came from different samples with different adaptors, could be exponentially amplified. Chromosome microdissection and microcloning cannot be an exception.

Acid Treatment Leads to Depurination One of the major concerns for the successful application of chromosome microdissection and microcloning technique was the extensive depurination of the chromosomal DNA caused by acid treatment during the sample fixation in chromosome preparation [ 14 ]. At present, depurination had been reduced or avoided by improving the chromosome preparation procedure [ 27 , 28 ]. Contamination PCR-mediated cloning of microdissected chromosome DNA made chromosome microdissection and microcloning more practicable.

However, it is difficult to completely avoid the DNA contamination from cytoplasm and foreign species [ 29 ]. Traditionally, the microdissection and microcloning steps should be carried out under the sterile condition as far as possible in order to effectively avoid the contamination from extraneous source DNA. The microdissection tools, the sterile water, enzyme mixture, the adaptor and the staining fluid should be filtered or treated under the ultraviolet light more than 30min.

The more chromosomes are microdissected, the more chance of contamination will be. Although flow cytometry can isolate more target chromosomes at one time, it may result in contamination from non-target chromosomes because of its sensitivity limitation.

Contamination could be reduced if only one microdissected chromosome or chromosome fragment is used to obtain microdissected chromosome DNA due to shortened manipulation time. For avoiding the contamination, Hu et al. Due to interference of sequences on homoeologous chromosomes in complicated allopolyploid genome, at present there is no way to sequence the allopolyploid genome, even one chromosome of the genome.

A direct approach to resolving this problem is to establish chromosome-specific DNA libraries using chromosome microdissection and microcloning. However, the present state of microdissection and microcloning technique cannot meet the needs for sequencing the complete specific chromosome DNA due to low coverage and small size of inserted DNA fragments. But the combination of mi-crodissection and microcloning with a recently developed technique, pclone, could be a potential way to solve the above-mentioned problems.

Zhang et al. They reported that the single cell E. By using phi29 polymerase and the N6 primer, the pclones lead to less amplification bias and larger insert fragment size

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