Physical mapping

Finding out the location of a gene on a chromosome is an important criterion for a sequencing project involving mapping studies. Accurately locating the genes on a chromosome helps to assign its function and adds value in linking them with each other. Thus we can find out whether the genes are present on the same chromosome. The relative position of the genes helps to construct a map and use it for analysis. A well-constructed map speeds up the genome sequencing task. A genome map, whether physical or genetic, is a guide that shows the position of the genes and their distinctive features. Just like a route map guides us throughout the road, a genome map guides in locating the genes and the possible changes in their composition. Knowing the genes and their function gives an estimate of the disease associated with the same. The chances of the relative errors are high without a map, especially when the DNA contains a repetitive sequence. The linkage groups showing the relative position of known genes associates the inheritance of two or more non-allelic genes.

Image 1: Physical mapping

The distance between the genes is an important part of mapping techniques. Linked genes are always situated close to each other. Their locations involve the same chromosome. Linked genes exhibit recombination frequencies. Their determination is the measure of their distance apart on the same chromosome. Significant technical advances in a genome project involve efficient mapping techniques. A subfield of genomics known as structural genomics is well-defined by three main techniques such as genetic mapping, physical mapping, and genome sequencing. The genome is divisible. Hence, dividing the genome into segments contributes greatly to mapping.

There are two types of mapping techniques. These techniques include genetic maps and physical maps. An inherited disease is located using a genetic map following the inheritance of a DNA marker present in the affected individuals. We can use genetic maps in finding the exact chromosome location of important disease genes. Hence a marker tracks a particular gene. The physical mapping does not rely on alleles. The genome sequencing project involving large genomes like human genomes relies mostly on the accuracy of a physical map. The reason behind requiring a physical map based genome project lies in the efficiency, accuracy, reliability, specificity and plethora of physical mapping techniques. A large number of eukaryotic progeny is unobtainable through a genetic map. The genetic mapping involves a relatively less number of meiotic events. Hence the gene mapping technique has a low resolution. The genetic maps with a limited accuracy may not perform well with human genomes. Physical mapping involves three main techniques such as restriction mapping, FISH, and STS mapping. The review mentions all the three types of physical mapping in details.

Image 2: Physical mapping technique 

1.   Restriction mapping:

The relative positions on a DNA molecule are easy to locate with the help of a restriction map. Though mapping using RFLPs on DNA markers locates the restriction sites that are polymorphic. Many sites remain unmapped. Especially non-polymorphic restriction sites are missed out. The only way to include these sites is to increase the marker density. In short, restriction mapping helps to achieve this target. A restriction map is nothing but a physical map of a piece of DNA showing recognition sites of specific restriction endonucleases. These enzymes recognize short DNA sequences. A key role of endonucleases is to protect the cells from viral attacks. Thus the endonucleases are mainly exploited while working with the DNA.

The basic methodology for constructing a restriction map

Treatment of DNA with one or few restriction enzymes is known as restriction digestion.  Eco RI and Bam HI are examples of frequently used restriction enzymes. Suppose we treat DNA with Eco RI, Bam HI and a combination of both the enzymes. Restriction enzymes can cleave small DNA fragments with predictable sizes. The electrophoretic technique separates the fragments based on the sizes. Two restriction enzymes help to cut the DNA in a double restriction. In case a fragment consists of two Bam HI sites, the original DNA is cleaved with Bam HI. A partial restriction product consists of uncut restriction sites since all the sites cannot be cleaved in one go.

It becomes difficult to measure several fragments of the same size. However, two classes of rare cutters may help to overcome this limitation. Some enzymes cut with seven to eight nucleotide recognition sequences. Other classes of enzymes recognize 5’-CG-3’ site. Sap I and Sgf I cut at seven or eight nucleotide sequences. However, the information regarding these enzymes is limited. A rare 5’-CG-3’ sequence in vertebrates follows methylation at the cytosine followed by deamination. Standard electrophoresis may not be able to separate DNA longer than 50 Kb. Orthogonal field alteration gel electrophoresis involves separation of DNA molecules up to 2Mb in length.

2.   Fluorescence in-situ hybridization (FISH):

The FISH technique helps to locate the position of a marker on a chromosome. It is a technique based on hybridization. It helps to identify specific chromosomes, chromosomal regions or markers through hybridization of fluorescently labeled probes. Fluorescence signals interpret the results. The DNA gets denatured into single strands for hybridizing with the probe. Mostly the fluorescent labels are non-radioactive. The fluorescent signal obtained from FISH measures its position relative to the short arm. Metaphase chromosomes are analyzed. Signals are detected using a fluorescence microscope. The fluorescent light detects the presence of a hybridized fluorescent signal. The absence of a fluorescent signal indicates the absence of chromosome material.

3.   STS Mapping:

A sequence tagged site is a known unique DNA sequence. STS is a small DNA sequence. STS mapping is accurate and reliable for mapping large genomes. Expressed sequence tags, random genomic sequences, and SSLPs are the sources of STSs. Expressed sequence tags are short unique DNA sequences derived from a cDNA library. ESTs are markers produced by PCR. Random genomic sequences are derived from random spanning of databases. SSLPs display length variations and are used as sequence tagged sites. Radiation hybrids and clone libraries are used as mapping reagents in STS mapping. The whole genome panels are preferred over single chromosome panels due to their efficient hybrid content.

References:
[1] Physical mapping of genes and genomes, Rekha Dixit, J. Biol. Engg. Res. & Rev., Vol. 1, Issue 1, 2014, 06-11 
[2] Molecular Biology, David P. Clark, Nanette J. Pazdernik

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