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DNA

Image : “DNA” by
PublicDomainPictures. License: CC0


Restriction Mapping

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:

  1. Multiple copies of a DNA segment are cut with restriction enzymes. A variety of different restriction enzymes are available for this purpose.
    Physical-Mapping

    “Restriction mapping provides physical maps of DNA fragments.” by Lecturio

     

  2. 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.
    Restriction-Mapping

    “Restriction Mapping.” by Lecturio

     

  3. 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.
    Restriction-Mapping-Step-3

    “Restriction-Mapping-Step-3.” by Lecturio

     

  4. This is how a physical map is constructed.

    Restriction-Mapping-Step-4

    “Restriction-Mapping-Step-4.” by Lecturio

Cytological Maps

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

Image: “Scheme of the principle of the FISH (Fluorescent in-situ hybridization) experiment to localize a gene in the nucleus.” by Mr. Matze. License: CC BY-SA 3.0

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

Sanger sequencing

Image: “The Sanger (chain-termination) method for DNA sequencing. (1) A primer is annealed to a sequence, (2) Reagents are added to the primer and template, including DNA polymerase, dNTPs, and a small amount of all four dideoxynucleotides (ddNTPs) labeled with fluorophores. During primer elongation, the random insertion of a ddNTP instead of a dNTP terminates synthesis of the chain because DNA polymerase cannot react with the missing hydroxyl. This produces all possible lengths of chains. (3) The products are separated on a single lane capillary gel, where the resulting bands are read by an imaging system. (4) This produces several hundred thousand nucleotides a day, data which requires storage and subsequent computational analysis.” by Estevezj. License: CC BY-SA 3.0

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.

  1. First, large DNA clones are isolated and arranged into contiguous sequences based on overlapping tagged sites.
  2. Large clones are then fragmented into smaller clones for sequencing.
  3. The entire sequence is assembled from the overlapping larger clones.

Shotgun sequencing: advanced computing

shotgun sequencing

Image: “In whole-genome shotgun sequencing (top), the entire genome is sheared randomly into small fragments (appropriately sized for sequencing) and then reassembled. In hierarchical shotgun sequencing (bottom), the genome is first broken into larger segments. After the order of these segments is deduced, they are further sheared into fragments, appropriately sized for sequencing.” by Commins, J., Toft, C., Fares, M. A. License: CC BY-SA 2.5

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.

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