Introduction
This article details preliminary work toward a collaborative project between the Laboratory for Structural Biology at the University of Alabama in Huntsville and the NASA Astrobiology Group at the Marshall Space Flight Center. The purpose of the collaboration is to characterize biological samples collected from extreme environments and thus obtain new insights on the biology of psychrophiles (i.e., microorganisms capable of thriving at temperatures as low as 0°C).
The moss specimen we have characterized here was found under 100m of ice in the Siberian permafrost and is estimated to be about 40,000 years old. A sample of this moss was grown successfully in culture. Our first objective was to amplify a DNA sequence from within the 18S rRNA gene and to compare the amplified sequence with known sequences in public gene banks.
Experimental Conditions
DNA Extraction: The moss sample was surface-sterilized with diluted ethanol followed by distilled water and ground under liquid nitrogen with a glass rod inside a 1.5ml tube. DNA was extracted using the plant tissue protocol provided with the Wizard® Genomic DNA Purification Kit (Cat.# A1120). See the Wizard® Genomic DNA Purification Kit Technical Manual #TM050 for protocol information.
DNA Amplification: Oligonucleotide primers were chosen within highly conserved sequences of the 18S rRNA gene from 20 different moss species. The PCR Core System (Cat.# M7660, M7665) was used for amplification. After 30 cycles, an aliquot of the amplification product was subjected to agarose gel electrophoresis.
PCR Fragment Purification: If electrophoresis showed a clean, single band, the remainder of the corresponding amplified DNA was purified using the Wizard® DNA Clean-Up System (Cat.# A7280). See the Wizard® DNA Clean-Up System Technical Bulletin #TB141 for protocol information. Otherwise the target PCR fragment was purified from a low-melting point agarose gel.
Results
The first PCR amplification yielded a single band of approximately 1kb, corresponding to the expected size of the 18S rRNA gene target sequence. However, possibly because of secondary structures interfering with the sequencing reaction, fewer than 150bp of sequence data could be obtained. Therefore smaller (<500bp) overlapping fragments covering the entire region of the 18S rRNA gene were amplified using a total of 14 different primers in various combinations. This strategy allowed us to obtain a consensus sequence of 1,817bp. The sequence was confirmed by subcloning two larger overlapping PCR products into a plasmid vector and sequencing the insert.
Characterization
The consensus sequence was compared to sequences available in the GenBank® (release 111.0, 15 April 1999) and EMBL (release 58.0, 15 February 1999) databases. It showed >99% homology with 15 different Bryophyta (moss) species, the best match being to Aulacomnium turgidum (accession number: AF023687) with 99.6% homology over the 1,781bp match. Classical taxonomy analysis suggested that the ancient moss may be Aulacomnium palustre, for which no sequence is currently available. This identification remains to be confirmed by sequencing the 18S rRNA gene from a modern sample of Aulacomnium palustre.
Summary
Several commercially available reagents were used to isolate, purify and amplify DNA from a 40,000 year old moss species originally found at a depth of 100m in the Siberian permafrost. The moss sample was grown in culture, and DNA was isolated using the Wizard® Genomic DNA Purification Kit. A segment of the 18S rRNA gene was amplified using the PCR Core System. Sequence analysis revealed that the DNA fragment exhibited more than 99% homology with 15 different Bryophyta (moss) species.