Physical map is a technique used in molecular biology 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 positions of the DNA bases. There are different techniques to visualize the gene location, including somatic cell hybridization, radiation hybridization and in situ hybridization. The different approaches to 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 a complete genome sequence in order to deduce any association between the target DNA sequence and phenotypic traits. If the actual positions of genes which control certain phenotypes are known, it is possible to resolve genetic diseases by providing advice on prevention and developing new treatments.
Low-resolution mapping Low-resolution physical mapping is typically capable of resolving DNA ranging from one base pair to several mega bases. In this category, most mapping methods involve generating a 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 those of mice and hamsters. The hybrid cell panel is produced by collecting hybrid cell lines containing human chromosomes, identified by polymerase chain reaction (PCR) screening with primers specific to the 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 producing 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 a process that coprecipitates human chromosome fragments with calcium phosphate onto the cell line, leading to a stable transformation of recipient chromosomes retaining human chromosomes ranging in 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 a radiation hybrid map by ordering the markers and breakpoints. This provides evidence on whether the markers are located on the same human chromosome fragment, and hence the order of gene sequence.
High-resolution mapping 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.
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