Re: After orchid DNA is extracted how can atomic weight be measured?

Orchid: Dendrobium sp.:  Introduction

Dendrobium are a genus of tropical Orchids that is comprised of
approximately 1200 species.  They are typically sought for their vivid and
various colours.  This has lead to the development of a variety of hybrids,
specifically of the Dendrobium nobile species.



Figure 1:  Orchid Dendrobium nobile, a species of orchid that
is commonly used in the production of hybrids.  Prized for their variety of
colours. 


The Answer:

Probably the simplest method to quantify genomic DNA is through photometry
(measurement with light).  Some examples are described under reference
(1).  Feulgen
reagent is commonly used to stain DNA within cells (thus removing the
necessity for purification) and the DNA can be quantitated using an
microspectrophotometer.  Both the reagent and equipment are likely to be
found in any histology group of the nearest university (perhaps, PGML, the Plant Genome Mapping
Laboratory at the University of Georgia).  Groups such as those at PGML may
also have suggestions on other techniques for further comparative genomic
analysis of the species you’re interested in.

 Purification of Plant Genomic DNA and analysis by Electrophoresis 

Determination of the molecular mass of genomic DNA from any multicellular
organism by electrophoresis is not a trivial task due to its great size. 
For example, the human genome has roughly five billion (5 x 10^9) base
pairs (bp).  Dividing by forty-six (the number of human chromosomes), still
gives an average of roughly 1.0 x 10^8 bp/chromosome.  Although many
species, plants included, can have smaller genome (such as Arabidopsis
thaliana with 125 million base pairs), all except microorganisms and
viruses are still too large for simple analysis by electrophoresis.

The most common application of agarose electrophoresis in molecular biology
is in the analysis of DNA pieces in the range of 100 - 10,000 bp.  You can
see from this, that common electrophoresis is not going to be very useful
for analysis of DNA pieces as large as those of a genome.

Such analysis is further complicated  by the fact that chromosomal/genomic
DNA of multicelluar organisms is notoriously difficult to purify intact due
to its immense size.  The large size of each piece means that it is
incredibly sensitive to sheering forces and will be very easily broken. 
Even in the most careful of cases, one can usually only hope to recover
fragments of the DNA that are 100,000 to 200,000 base pairs (see reference
3 for a discussion).

Therefore, a determination of the purified genomic or chromosomal molecular
weight by electrophoresis is probably not a viable option and you will
likely have greater success using the photometric technique described earlier.

Resources and references

1. 

An improved acriflavine-Feulgen reagent for quantitative DNA
cytofluorometry by JW Levinson, RG Langlois, VM Maher and JJ McCormick


Feulgen banding of Heterchromatin in Plant Chromosomes.  A free article
from the Journal of cell science.  It’s a little old now (1983) but should
still give a good idea of what’s involved.


Chapter 5.1  Quantifying the Genome.  This is an online chapter in Lee
M. Silver’s ‘Mouse Genetics:  Concepts and Applications’ but it gives a
good overview of the general ideas.

2. 

Plant Genome Mapping Laboratory, University of Georgia

3.  

Isolation and Gel analysis of DNA from Plants a PDF instruction manual
from Edvotek (such instruction manuals
are often a great source of general information).