Locate candidate clones

At present, the focus of human genome research is shifting from "structure" to "function", a so-called "post-genomics" (functional genomics) era with genomic function research as the main content is about to arrival. How to obtain the functional information of genes, that is, the genetic information related to major human diseases and important physiological functions, is before us. Positional candidate cloning strategy (positional candidate cloning) is an improvement and development of traditional positional cloning, and is increasingly valued for its effectiveness in cloning disease-related genes.
The Human Genome Project has identified a series of molecular markers distributed on 24 chromosomes throughout the genome. According to the sequence and location information of these molecular markers, combined with hybridization or PCR methods can quickly detect hot spots on chromosomes related to tumors or genetic diseases, and then clone disease-related genes from them. Candidate cloning overcomes the tedious and slow drawbacks of classical location cloning, which purely relies on linkage analysis for chromosomal location, greatly speeds up the process of cloning work, and it is not limited to genetic diseases. Work on cloning of sense genes. The genetic map started to be constructed internationally in 1994 and first published in 1996 is a combination of physical maps marked with cDNA-based STS (sequence-tagged site) and genetic maps marked with polymorphic microsatellites. It is a map The aspect makes the chromosome positioning more accurate, reliable and precise, and at the same time provides great convenience for obtaining disease-related candidate genes from the candidate region, making the cloning of the target gene easier and faster, which injects into the development and application of this method Greater vitality is manifested by the subsequent cloning of 16 new disease-related genes, such as the tumor suppressor gene DPC4 isolated from pancreatic cancer. In October 1998, GeneBank released the latest "Gene Map 98", which has more information, and has greatly improved accuracy and precision. It contains 41 464 STS markers, representing 30 181 genes. Location information, that is, about half of the 60,000 to 70,000 genes in the entire human genome has been completed [2]. In short, with the advancement of the Human Genome Project, locating candidate clones has become an effective method for cloning disease-related genes.
Mapping candidate clones includes three basic steps: genome mapping, candidate cDNA acquisition, full-length and functional analysis. This article provides an overview of the development and application of this strategy.

1. Genomic location

Newly developed, especially in the isolation of tumor susceptibility genes, is to use PCR to detect the high frequency of loss of heterozygosity (LOH) or homozygosity in multiple samples to obtain the location of disease candidate genes [3] . Somatic tumor suppressor gene inactivation is a major factor leading to canceration, the most common manifestation is the loss of heterozygosity. Frequently, the region of high frequency of loss of heterozygosity on the chromosome contains one or more tumor suppressor genes. Using molecular markers (including STS, microsatellite markers, etc.) scattered on each chromosome, PCR is used to detect the deletion of chromosomes in multiple tumor samples, and the high frequency of LOH can be determined, that is, the growth of related tumors The chromosomal location of the negative regulatory factor can be accurate to the area indicated by the molecular markers of the genome, which can further be used for gene cloning. Using this method, we have successfully cloned several tumor suppressor genes, such as BRCA1, BRCA2 in breast cancer and DPC4 gene in pancreatic cancer. At the same time, the continuous development and promotion of FISH technology, the maturation of chromosome microdissection technology, can cut specific chromosome bands under the microscope, prepare chromosome band specific probes, perform FISH analysis on normal and diseased tissues, and compare and determine diseases Chromosome banding of related genes. In recent years, the establishment of the RH spectrum (radiation hybrid map) has made it possible to precisely locate chromosomes. These methods are faster and more accurate than the original linkage analysis. For example, it took LC Tsui about 10 years to use linkage analysis to locate the cystic fibrosis gene on chromosome 7. Now, it may take only one year or less to complete the work.

2. Screening of candidate cDNA

The information provided by genome mapping contains certain molecular markers, and the corresponding clones can be screened directly from the YAC (yeast artificial chromosome) library, or from the BAC (bacterial artificial chromosome) library, PAC (P1 artificial chromosome) library by PCR or hybridization. . The chimerism of YAC library is serious and the operation is more difficult. It has been gradually replaced by PAC library and BAC library. The PAC system is a cloning system based on bacteriophage P1. It can accommodate inserts of 70 to 100 kb and can selectively distinguish recombinants from non-recombinants, and has two replication mechanisms at the same time. Single-copy replicons can be used for stable clonal proliferation, while multi-copy replicons can be used for DNA preparation under the control of the Lac operon [4]. The BAC system is based on a vector system derived from E. coli that can accommodate genes in large capacity and has good stability. It can accommodate inserts over 100kb. The average length of BAC cloned inserts is 120kb, and the maximum can reach about 240kb. [5]. Good stability and ease of operation are the main advantages of the BAC system. Starting with these clones, candidate cDNAs are obtained using methods that rely on appropriate cDNA libraries, methods that rely on characteristic sequences, or methods that rely on expression properties.
(1) Methods that rely on an appropriate cDNA library include direct screening and cDNA selection. The direct screening method uses genomic DNA to directly select the cDNA located in the genomic fragment from the cDNA library, such as separating foreign fragments from YAC [6], PAC [7], BAC clones covered with candidate regions as probes from the library Candidate genes are screened in the middle; and the cDNA selection method [8] is exactly the opposite of the direct screening method, which fixes genomic DNA on the membrane, hybridizes it with total cDNA, and recovers cDNA fragments that can be combined with genomic fragments by PCR. The advantage of the direct screening method is that it avoids the subcloning operation of large fragments of candidate regions, and the information of multiple transcripts can be obtained in a single screening. However, the disadvantage is that it is difficult to purify large fragments, and the relatively convenient purification of PAC and BAC clone fragments has become the trend of this method. At the same time, due to the complexity of the probe, the hybridization background is deepened and false positives are increased. It is easy to ignore short exons or low abundance cDNA. The greatest advantage of the cDNA selection method is the intervention of the PCR reaction, which greatly improves the sensitivity of selection and can detect some rare transcripts.
(2) The method of relying on characteristic sequences is to directly separate cDNA fragments from genomic DNA according to the sequence characteristics inside and on both sides of the transcript, mainly including CpG island search method, exon trapping method, cross species sequence Source comparison method (cross-species sequence homology), Alu PCR method and direct sequence analysis method. CpG islands, also known as HTF islands, are small areas rich in GC. Most of the transcripts (usually in the 5 ′ upstream region) contain unmethylated CpG islands [9]. Using some rare enzymes that recognize CpG islands, such as SacⅡ, BssHⅡ, EagⅠ, etc., genomic DNA is cut to obtain sequences located near CpG islands as probes to obtain transcripts from total RNA or cDNA libraries. The exon capture method is based on the identification of functional 5 ′ and 3 ′ splice sites on both sides of the exon to isolate exon fragments from genomic DNA [10]. The basis for comparing the homology of cross-species sequences is that the coding region sequence is much more conservative than non-coding regions in different species [11], and the DNA of the candidate region is divided into multiple subclones, which are respectively A DNA clone that hybridizes and has a positive signal with DNA from different species may be the coding region gene. This method relies on sequence homology at the DNA level between species. Alu sequences are short repetitive sequences scattered in the human genome. By designing human-specific Alu sequences as primers, PCR-based methods can be used to quickly obtain specific sequences of human origin from YAC, BAC, PAC clones or human and mouse hybrid cells [12]. The direct sequence analysis method uses GRAIL and GeneScan and other software to analyze and speculate the coding gene in the genome sequence information [13].
(3) Expression-dependent method, that is, Northern hybridization analysis method [14]. The genomic DNA of the candidate region is used as a probe to hybridize with total RNA, a positive band is cut, reverse transcribed into cDNA and cloned, and a transcript located in this genomic fragment can be obtained.
Each of the above methods has its own advantages and disadvantages. It is necessary to select an appropriate combination of one or more methods to achieve the research purpose according to the raw materials and the purpose to be achieved. In addition, there are many other methods, such as microdissection and microcloning, subtractive cDNA hybridization, and recombinant screening.

3. Full length and functional analysis

After obtaining a large number of candidate cDNAs, clone the full-length gene by screening cDNA libraries or RACE. In order to determine the pathogenic genes, it is necessary to detect their changes in the affected families one by one and analyze their functions. Functional analysis is often a difficult point in locating clones, because different diseases have different characteristics, which requires different functional detection systems for analysis. Commonly used are studies on changes in cell morphology and function after the expression of sense or antisense genes at the cellular level. In tumor studies, changes in cell morphology and contact inhibition, changes in growth capacity of soft agar, and tumorigenicity analysis in nude mice are examined. Further functional analysis includes individual-level gene knockout experiments.
Now some new ideas have been developed in combination with other methods, such as combining with subtractive hybridization to screen differentially expressed genes located in candidate regions, greatly increasing the possibility of obtaining valuable genes. At the same time, with the development of the transcription map [15] in the Human Genome Project, many ESTs have been accurately located on different regions of chromosomes, and functional studies can be conducted directly. With the deepening of research, more and more EST mapping work has been completed, and the gene transcription map has been continuously improved. We can skip the second step of positioning cloning and go directly to functional identification and full-length gene cloning. This may be the fastest way to locate clones. As analysis and isolation methods continue to improve and the Human Genome Project is being implemented vigorously, various new ideas and methods are emerging in an endless stream, and the cycle of positioning cloning methods will continue to shrink, providing the possibility for humans to eventually clone various disease genes.

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