Physical mapping

Physical mapping is a molecular biology technique to find the order and physical distance between DNA base pairs by DNA markers. It is one of the gene mapping techniques which can determine the sequence of DNA base pairs with high accuracy. Genetic mapping, another approach of gene mapping, can provide markers needed for the physical mapping. However, as the former deduces the relative gene position by recombination frequencies, it is less accurate than the latter. Physical mapping is a molecular biology technique to find the order and physical distance between DNA base pairs by DNA markers. It is one of the gene mapping techniques which can determine the sequence of DNA base pairs with high accuracy. Genetic mapping, another approach of gene mapping, can provide markers needed for the physical mapping. However, as the former deduces the relative gene position by recombination frequencies, it is less accurate than the latter. Physical mapping uses DNA fragments and DNA markers to assemble larger DNA pieces. With the overlapping regions of the fragments, researchers can deduce the position of the DNA bases. There are different techniques to visualize the gene location, including somatic cell hybridization, radiation hybridization and in situ hybridization. Different approaches of physical mapping are available for analyzing different sizes of genome and achieving different levels of accuracy. Low- and high-resolution mapping are two classes for various resolution of genome, particularly for the investigation of chromosomes. The three basic varieties of physical mapping are fluorescent in situ hybridization (FISH), restriction site mapping and sequencing by clones. The goal of physical mapping, as a common mechanism under genomic analysis, is to obtain complete genome sequence for deduction of any association between the target DNA sequence and traits. If the actual position of genes which control certain phenotype is known, it is possible to resolve genetic diseases by providing advice on prevention and developing new treatments. Low-resolution physical mapping is typically capable to resolve DNA ranging from one base pair to several mega bases. Under this category, most mapping methods involve the process of generating somatic cell hybrid panel, which is able to map any human DNA sequences, the gene of interest, to specific chromosomes of animal cells, such as mice and hamsters. Hybrid cell panel is produced through collecting hybrid cell lines containing human chromosomes, identified by polymerase chain reaction (PCR) screening with primers specific for human sequence of interest as the hybridization probe. The human chromosome would be presented in all of the cell lines. There are different approaches to produce low-resolution physical mapping, including chromosome-mediated gene transfer and irradiation fusion gene transfer which generate the hybrid cell panel. Chromosome-mediated gene transfer is the process that coprecipitating human chromosome fragments with calcium phosphate onto the cell line, leading to a stable transformation of recipient chromosomes retaining human chromosome of the size from 1 to 50 mega base pairs. Irradiation fusion gene transfer produces radiation hybrids which contain the human sequence of interest and a random set of other human chromosome fragments. Markers from fragments of human chromosome in radiation hybrids give cross-reactivity patterns, which are further analyzed to generate radiation hybrid map by ordering the markers and breakpoints. This provides evidence on whether the markers are located on the same human chromosome fragment, thus the order of gene sequence. High-resolution physical mapping could resolve hundreds of kilobases to a single nucleotide of DNA. A major technique to map such large DNA regions is high resolution FISH mapping, which could be achieved by the hybridization of probes to extended interphase chromosomes or artificially extended chromatin. Since their hierarchic structure is less condensed comparing to prometaphase and metaphase chromosomes, the standard in situ hybridization target, a high resolution of physical mapping could be produced. FISH mapping using interphase chromosome is a conventional in situ method to map DNA sequences from 50 to 500 kilobases, which are mainly syntenic DNA clones. However, naturally extended chromosomes might be folded back and produces alternative physical map orders. As a result, statistical analysis is necessary to generate the accurate map order of interphase chromosomes. If artificially stretched chromatin is used instead, mapping resolutions could be over 700 kilobases. In order to produce extended chromosomes on a slide, direct visual hybridization (DIRVISH) is often carried out, that cells are lysed by detergent to allow DNA released into the solution to flow to the other end of the slide. An example of high resolution FISH mapping using stretched chromatin is extended chromatin fiber (ECF) FISH. The method suggests the order of desired regions on the DNA sequence by analyzing the partial overlaps and gaps between yeast artificial chromosomes (YACs). Eventually, the linear sequence of the interested DNA regions could be determined. One more to note is that if metaphase chromosome is used in FISH mapping, the resolution resulted will be very poor, which is to be classified to low-resolution mapping rather than a high-resolution mapping.