Biology 475

Molecular Biology

Lab Five and Six - DNA Sequence Analysis

Rogan Rattray

copyright 2003

Introduction

These labs used DNA samples that had been processed using large-scale plasmid isolation and use the 16S sequences that have been inserted into the plasmid after PCR, as templates for replication. Unlike conventional replication however, the complementary strands that are being generated will be shorter partial sequences that will end in a Di-Deoxynucleotide at the 3’ end which will not allow for further addition of nucleotides to that sequence. The process is repeated many times until many partial sequence copies exist. These copies will then be separated by acrylamide gel electrophoresis.

	DNA Sequencing Methods
Reaction Set-up Methods: The red tubes in the picture (right) were set-up with either the fluorescent forward or reverse primers. Reaction buffer and Taq Polymerase were also added to each red tube. The contents of the red tubes were then split equally between two purple tubes containing sample DNA. Two purple tubes were for sample plus forward primer the other two were sample and reverse primer. The contents of the purple tubes were then added to individual strip tubes containing nucleotides plus either A, T, G or C Di-Deoxynucleotide bases (not shown).
Gel Pouring Methods: The Polyacrylamide gel components were mixed and poured between the cleaned and assembled glass gel plates (right). The solid end of the comb was placed into the top of the gel before it polymerized so as to prepare a loading well.

DNA Sequencing Results

Sample 49 Forward aTATAGGgcGAATTGGGCCTCTAGATGATGCTTGAgcGGCGCAGTGTGATGGATATCTGC

AGAATTCGCCCTTGACGGGCCCTCGCAGGCGACGACGTTTCCGCCGTCTGCACCGTCATG

TCCAGTCTGGGTAAGGTTCTGCGGGTTGCGTCGAATTAAACCACACGCTCCGCTGCTTGT

GCGGGCCCCCGTCAATTCCTTTGAGTTTTAAGcTTGCGCTCGTAGTTCCCAGGCGGACCA

CTTATCGCGTGAGCTAGGGCGCGCCGCGACGTCAATAGTCGCCACACGCCGAGTGGTCT

CGTTTACGGCGTGGACTACCCGGGTATCTAATCCGGTTTGCTCCCCACGTGTCGCGCCTC

AGCGTCAGCTGCGGCCAGCCCCCTGGcTTCCCCCTTGGTCTTCCTGCCGATCTcTACGgA

TTTcACCgCTACACCGGCAATTCGAGGGGCCTCTcCGCAGCTCGAGCGACCCAGTTTGCC

ATGGCCTCCCCCGGTTGAGCCGGGGGCTTTACAcGACACTAAGGTTGCCGCCTGCGCGCG

CTTTACGgCCAGTGACTCGGACAACGcTCgCCCCCTCTGTTTACCGGGGTGCTGGCACAG

AGTTAGcCGGGGCTTgTTCCGGGGGTACCGTATATtGTCCCCC 

Sample 49 Reverse ATGaTGATTAGCAAGCTTGGTACGAGcTCGGaTCACTAGTAACGGCGccAGTGTGcTGGA

ATTCGCCCTTGTGGCGCACGGCTGAGTAACACGTGGGAACCCGCCCCCCGGTGGGGGGaT

AACGcGACGAAAGTTGCGCTAATCCCGCATACGTCCTTCGGGGGAAAGCGCAGTGCGcGc

CGGGGGAGGGGCCTGCGGCCaTCAGGTCGTTGGTGGGGTAAGGGCCTACCAAGCCGATGA

CGGGTAGCTGGTCTGGGAgGGATGACCAGCCAGACTGGGACTGAGAcAaGGCCCAGACTc

CTACGGGAGGcAGCAGCAAGGAATTTTCGGCAATGGGCGCAAGCTGACCGAGCAACGCGC

GTGCGGGATGACGGCCTTCGGGTTGTAAACCGCTTTTCGGGGGGACGTGATGACGGTACC

CCCGGAACAAGCCCCGGCTAACTTGTGCCAGcAGCCGcGGTAAGACAGAGGGGGCGAGCG

TTGTCCGGAGTCACTGGGCGTAAAGGCGcGcAGGGGCAACCTTAGTGTCGTGTGAAAGCC

CCCGgTTAACCGGGGGAGGcCATGGC 

DNA Sequencing Results

Top Five Different BLAST Species

(Using forward sequence only)

AF421730
Chloroflexi;Chloroflexaceae;

Appl. Environ. Microbiol. 68 (1), 346-355 (2002)

Molecular Characterization of Novel Red Green Nonsulfur Bacteria
from Five Distinct Hot Spring Communities in Yellowstone National Park
Boomer,S.M., Lodge,D.P., Dutton,B.E. and Pierson,B.

UBA421656

uncultured bacterium

Appl. Environ. Microbiol. 68 (9), 4593-4603 (2002)

Microscopic examination of distribution and phenotypic properties
of phylogenetically diverse Chloroflexaceae-related bacteria in hot spring microbial mats
Nubel,U., Bateson,M.M., Vandieken,V., Wieland,A., Kuhl,M. and
Ward,D.M.
 
M62775
Firmicutes;Eubacterium.
Nature 345 (6270), 63-65 (1990)
16S rRNA sequences reveal numerous uncultured microorganisms in a natural community
Ward,D.M., Weller,R. and Bateson,M.M.
 
AB041226
Roseiflexus castenholzii
Int. J. Syst. Evol. Microbiol. 52 (Pt 1), 187-193 (2002)
Roseiflexus castenholzii gen. nov., sp. nov., a thermophilic,
filamentous, photosynthetic bacterium that lacks chlorosomes
Hanada,S., Takaichi,S., Matsuura,K. and Nakamura,K.
 
AB079641
Kouleothrix aurantiaca
Water Science and Technology (2002) In press
Characterization of Type 1851 organism isolated from activated sludge samples.
Kohno,T., Sei,K. and Mori,K.

Discussion

Since we compared the sequences that we obtained from our samples to other 16S sequences on file with GenBank, we can note what kinds of species our samples may potentially represent. We could take these comparisons a step further and generate a phylogenetic tree of our unknown samples and other select samples. We would want to use know sequences from some species that we assume are closely related to our sample. We would also want to use some species that we figure to be more distantly related to our sample and see just where ours would fall in among these. These kinds of analyses can help us determine whether theories about the identity of our sample will hold, or they may point us in a different direction as to how these Red Layer hot spring mat bacteria should be categorized.