Table of Contents
Physical mapping allows us to find the actual physical location of each gene on the chromosome—all the way down to such a granular level 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 segment of DNA are cut with restriction enzymes. A variety of different restriction enzymes are available to use 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.
In order to create genetic maps, it is necessary to use labeling and tags, so the result might resemble these cytological maps. The chromosome has been broken down into sections, which show the physical locations of the genes. Cytological maps use staining in order to mark places on the genome, thus allowing for a whole view of each chromosome and therefore, the entire genome.
FISH – Fluorescence In-Situ Hybridization
FISH is one of the staining techniques used to mark chromosomes with fluorescent dyes and see if they contain particular genes. The primary purpose of the cytological maps is to characterize 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 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, with electrophoresis separating the resulting pieces by size. Each clone, provides different pieces of DNA, which can be aligned because of the STSs.
In order to identify these STSs, polymerase chain reaction (PCR) is used with probes; 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 say exactly where a gene is located on a chromosome by 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
Methods that can be used in genome sequencing can be broken into two categories: the clone-by-clone sequencing method and the shotgun sequencing method.
Clone-by-clone sequencing: physical mapping
Also known as BAC to BAC (because we are putting it from bacterial artificial chromosome to bacterial artificial chromosome), or hierarchical sequencing. It requires a little less lining up of fragments.
- As a first step, large DNA clones are isolated. These are 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
It’s also known as whole genome shotgun sequencing, and is only possible because of the higher levels of computing that are possible these days. Rather than doing BAC to BAC, we just hack up the entire genome into millions of little pieces. Previously, this method consumed a lot of computer resources, but nowadays, with the advantage of powerful computing, it is the better option.
- Cut DNA of entire chromosome into small fragments and clones.
- Sequence each segment and arrange based on overlapping nucleotide sequences.
Popular Exam Questions
1. All of the following statements regarding restriction mapping are true, except:
- restriction enzymes cut multiple copies of a segment of DNA.
- physical maps provide actual physical locations of genes.
- physical mapping uses landmarks within DNA.
- the larger fragments will go more distance through the gel.
2. All of the following statements about restriction enzymes are wrong, except:
- DNA sticky ends are always the end result of restriction enzyme cutting.
- restriction enzymes are considered endonucleases.
- restriction enzymes are considered exonucleases.
- the defense system of the bacteria does not include restriction enzymes.
3. Different methods may be used in genome sequencing, including:
- clone-by-clone sequencing.
- shotgun sequencing.
- Both a and b.
- None of the above.