Monday, 2 March 2020

Molecular Analysis of DNA

 DNA Molecular Analysis image


DNA products can be obtained from living plant and animal cells quite easily. This molecular analysis of DNA is related to genetics and then analyzed to determine evolutionary relationships among individuals representing various taxonomic groups. DNA can also be removed from dried plant samples and frozen plant and animal tissues, usually causing degradation and therefore loss of systematic value.


Protein electrophoresis

Because proteins vary in size and have both positive and negative charges on their surface, proteins migrate differently in a starch or acrylamide gel to which an electric field is applied. Proteins separated by an amino acid substitution will exhibit different positions on a gel. This substrate is visualized using an enzyme of specific interest and stain upon which the enzyme reaction occurs. Polyphoresis is used to determine the genotype of individuals in polymorphic populations. Allozyme electrophoresis identifies allozymes, forms of an enzyme coded for by different alleles at a locus. Isozyme electrophoresis is similar but isozyme is products of different loci in a single genome.


DNA-DNA hybridization

This method basically involves melting double-stranded DNA of two samples at high temperatures and then lowering temperatures to reanneal the single strand from each sample. Reannealed homoduplexes are fairly tightly bound, and relatively high temperatures are required o melt them; however, the more unrelated the two were original, the melting of the reannealed heteroduplexes requires lower temperatures. This technique has been used in animal studies.

Restriction Fragment Length Polymorphism (RFLP)

Basically, this method takes a molecule of nuclear DNA and cuts it at known points using various restriction enzymes that are specific for short ( usually four or six ) sequences of base pairs. Fractions resulting from this cutting are electrically isolated to produce genetic markers that can be used to make different population comparisons. Visualization of this fragment can be accomplished by radioactive labeling or staining. Direct sequencing of the fragments can also be done if they prove systematic value.

Randomly Amplified Polymorphic DNA (RAPD)

This molecular analysis of DNA technique basically uses short and inverted repeating short synthetic oligonucleotide primers that scan a genome and then sequence between using PCR (polymerase chain reaction) to make multiple copies. As the segment of DNA is being amplified, and the random length (its sequence is not usually known), the sizes (lengths) of these fragments resolved on gels often vary from genotype to genotype. RAPD has become especially useful in population genetics because it can generate genetic markers that are polymorphic among individuals in the same population, so " genetic individuals " can be recognized in clonal species like fungi. It has also been used to produce "genetic fingerprints" to resolve legal disputes. RAPD is often chosen quickly and reliably with other molecular techniques and at relatively low cost.

Direct Sequencing of Genomes

It involves the precise determination of the nucleotide sequence of the genome. Although this is more laborious, it provides relatively reliable data with which to establish a phylogenetic position of related taxa. Choices of the genome can affect the results obtained, but on the whole, sequencing is used to establish monophyly or polyphyly of groups, ancestral vs derived when the phylogenetic analysis is the object of the study.

As a general rule, choice of DNA sequences to be obtained are based on the amount of evolutionary time involved in producing the variations in the group under study (More conserved parts of the genome are used when large amounts are involved).

The mitochondrial genome (mtDNA) in animals is often sequenced for comparison with the same sequence; This is because mtDNA occurs maternally, not subject to recombination and has numerous known orthologous genes.

Few generalizations are:


1. The molecular analysis technique can be applied to a number of distinct genomes (nuclear, mitochondrial, chloroplast) using a number of different nucleic  acids (rDNA, cpDNA, various RNAs, etc.)

2. Molecular clocks do not keep perfect time. Every gene appears to change at a rate independent of others. Also, different organisms exhibit different rates for the same genes and organisms in the same clade may exhibit different rates at different times.

This is an area in which phylogenies can vary widely depending on different techniques. For example, consider the concept of "mitochondrial Eve", a paleolithic female who lived 200,000 years ago in Africa and from whom all present-day humans descended. This based on mitochondrial DNA Molecular Analysis, Which is inherited exclusively through the female and modified only by mutation; it also uses a standard estimate of the mutation rate of 2-4% per million years. These origins have been traced by paleontologists who use fossil and cultural evidence to track human origins, considering the inclusion of several sites in Asia, Africa, and Europe.

Assumptions


Rate of molecular change is constant

The clock can be calibrated using a reliable secondary source of information (fossils, for example). The psychiatrist changes the rates at different times.

DNA Molecular Analysis 

In addition to proteins, DNA is often analyzed directly as a source of similarities between organisms.

Systematics Molecular Analysis

Use of molecular data as characters to assess the phylogenetic position of taxa for classification. It generally uses sequence data from specific genes (rDNA, mtDNA, cpDNA, etc.). Sequence comparisons reveal patterns of divergence in the past.

The chloroplast DNA of plants changes very slowly and can be used to establish phylogenies at the phylum level as the origin of Angiosperms, relationships among gymnosperms, and origin of land plants.

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