Saturday, 1 April 2017

Proteinase K, can it improve the quality of DNA extracted from Sulfhydryl rich leaves ?

By Timothy Macqueen

I have recently begun work on attempting to extract DNA from the leaves of Protea susannae and P. eximia hybrids using the Doyle&Doyle CTAB method. There have been several set-backs in the quality of the DNA I have extracted. In many of the DNA gels I have run, only one or two of my samples have shown successful amplification. 
An early gel run. The only amplified DNA sample was the second to right. The rest were PCR and gel positives


 The 260/230 ratios of my samples has been consistently low. I wanted to determine the reason for this, so I posted my problem on the scientific community website, Researchgate. On it, H. Ayyez of the University of Al-Qadisiyah suggested that this was because I was experiencing organic contamination due to not adding Proteinase K as a part of my extraction. 

Before I continue, I'd better explain the importance of the 260/280 and 260/230 values to DNA amplification and analysis. A Nano-drop is used to measure these values. The 260/280 value of a DNA sample indicates the purity of the DNA extracted. A value around 1.8 is considered to be pure. 

Low 260/280 ratios may be caused by: 
• Presence of residual phenol or other reagents associated with the extraction protocol
• A very low concentration( > 10 ng/ul).of nucleic acid

The 260/230 ratio is important as a second measure of DNA purity after the 260/280 ratio. An optimum value for this ratio is between 2.0-2.2. 

Low 260/230 ratios may be the result of:
 • Carbohydrate carryover (often a problem with plants). 
• Residual phenol from nucleic acid extraction.  OR most importantly-

  • The presence of organic contaminants, such as (but not limited to): phenol, TRIzol, chaotropic salts and other aromatic compounds.                                                                                                                                                                                                       Samples with 260/230 ratios below 1.8 are considered to have a significant amount of these contaminants that will interfere with downstream applications. This is especially true for reverse transcription.
    Protea hybrid at Van Staadens nature reserve. It contains many of the distinct morphological traits of  P. susannae. That distinct sulphurous odor, distinctive to leaves containing sulfhydryl compounds. Could these organic compounds be the cause for all my troubles trying to amplify the DNA of my hybrid proteas?    

One such compound is a Thiol. This is a organosulphur compound that contains carbon bonded sulfhydryl group. This is known to occur in my samples because the leaves of P. susannae and its hybrids when crushed, produce a distinct sulfurous odor. This odor is distinct  to Thiols. 


The 260/280 values in my samples were very close to or at 'pure' ratio values while my 260/230 (yellow highlighting) ratios were very low. 
So what can Proteinase K do to improve the quality of my 260/230 ratios? Well, Proteinase K is a serine proteinase which is able neutralise a wide variety of contaminating proteins from organic compounds. It is also used to inhibit nucleases from degrading the nucleic acids to be extracted. 

Will it work? Well, the only way I can know for certain is by testing this hypothesis. Currently our lab is out of stock of Proteinase K. We have just ordered a new batch though. Once it is received I will use it in my extraction method and report back to this blog on whether or not it improved the quality and purity of my DNA extractions. 

References


Cremlyn, R.J. 1996. An Introduction to Organosulfur Chemistry. Chichester: John Wiley and         Sons. ISBN 0-471-95512-4.
Patai, S. 1974. The chemistry of the thiol group. London: Wiley. ISBN 0-471-66949-0.

Links to website references (All accessed on 01/04/2017)






1 comment:

  1. Proteinase K (PROK) is a serine protease with broad specificity towards aliphatic, aromatic and other hydrophobic amino acids. PROK has a molecular weight of 27,000 daltons and is Ca2+ dependent. It is not inactivated by metal ion chelating agents such as EDTA, Proteinase K

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