Table of Contents
Physical mapping allows us to find the actual physical location of each gene on the chromosome to such a granular level that we know precisely at what letter a gene begins.
- Physical mapping uses landmarks within DNA.
- Genetic maps provide the relative location of the genes, based on recombination frequency.
- Physical maps provide the actual physical locations of each gene.
Restriction mapping provides physical maps of DNA fragments. The process is performed in the following steps:
- Multiple copies of a DNA segment are cut with restriction enzymes. A variety of different restriction enzymes are available for this purpose.
- The fragments produced by enzyme A only, enzyme B only, and by enzyme A and B together, are run side-by-side on a gel. The negatively charged DNA runs toward the positive pole and, since the larger fragments will move less distance through the gel, this procedural step separates the fragments by size.
- Thus, the fragments are arranged so that the smaller ones (produced by the simultaneous cut of enzymes A and B) can be grouped to generate the larger ones (produced by the individual enzymes). These pieces are taken and compared according to their sizes and lengths.
- This is how a physical map is constructed.
Labeling and tags are necessary to create genetic maps, so the result might resemble these cytological maps. The chromosome has been broken down into sections that show the genes’ physical locations. Cytological maps use staining to mark places on the genome, allowing for a complete view of each chromosome and, therefore, the entire genome.
FISH – Fluorescence In-Situ Hybridization
FISH is a staining technique for marking chromosomes with fluorescent dyes to see if they contain particular genes. These cytological maps are helpful for characterizing chromosomal abnormalities.
Sequence Tagged Sites (STSs)
STSs can provide a sort of scaffolding that shows how the pieces in the genome go together. This helps to investigate the locations of known DNA sequences on a chromosome. STSs comprise of 200 to 500 base pair sequences that have a single occurrence. DNA fragments from DNA libraries are cut with restriction enzymes and run on the gel. Electrophoresis separates the resulting pieces by size. Each clone provides different pieces of DNA, which can be aligned because of the STSs.
Polymerase chain reaction (PCR) is used with probes to identify these STSs. The probes will attach when the DNA separates; then, they can be located by using visualization techniques.
The Ultimate Physical Map: The Sequencing of the Entire Genome
The ultimate physical map represents the exact DNA sequence on a chromosome. We can pinpoint a gene’s location on a chromosome using DNA sequencing. Vectors containing cloned DNA from libraries can be used to sequence a genome.
Sanger Sequencing: The Enzymatic Method
Dideoxynucleotides are a critical component. 3′ OH is needed for DNA polymerase to add new nucleotides.
- DNA replication in vitro
- Termination of replication occurs every time a dideoxynucleotide shows up.
Genome Sequencing: The Development of Artificial Chromosomes
There are two types of genome sequencing methods: the clone-by-clone sequencing method and the shotgun sequencing method.
Clone-by-clone sequencing: physical mapping
This method is also known as BAC to BAC (because we are putting it from bacterial artificial chromosome to bacterial artificial chromosome), or hierarchical sequencing.
- First, large DNA clones are isolated and arranged into contiguous sequences based on overlapping tagged sites.
- Large clones are then fragmented into smaller clones for sequencing.
- The entire sequence is assembled from the overlapping larger clones.
Shotgun sequencing: advanced computing
This method is also known as whole-genome shotgun sequencing. It is possible because of better computer technology.
Rather than hierarchical sequencing, which is time-consuming, the shotgun sequencing method breaks up DNA sequences into randomly sized small pieces and then reassembles the sequence by looking for regions of overlap.
Improved computing technology and software make shotgun sequencing faster and more efficient than BAC to BAC sequencing. However, this method is most effective when there is a reference genome for matching. Otherwise, it is prone to errors that need to be corrected with more labor-intensive types of sequencing.