Biology 475 Molecular Biology Lab Five and Six - DNA Sequence Analysis Shanna Briggs Copyright 2003

Introduction The goal of this lab period was to sequence the DNA inserts from the clone plasmids isolated during lab four. I sequenced inserts from clones numbers 81 and 82.

	DNA Sequencing Methods
Reaction Set-Up Methods: Each tube contains the ddNTP labeled on the tube, all four dNTPs (in the buffer), DNA, a single primer, and thermal stable polymerase. The samples were run in the Thermal Cycler with the “sequence program.”
Gel Pouring Methods: To “pour” the gel, we layed it on an incline. David used a syringe to slowly and steadily inject the acrylamide between the two glass plates. I gently tapped on the glass to make sure that the gel flowed in an evenly and did not form any bubbles.

	DNA Sequencing Results
Sample 81, Forward.	TATAGGGCGAATTGGGCCTCTAgATGCTCGAGCGGCCGCCAGTGTGATGGATATaTGGAGAATTGgcCTTAaGG GAGGGAGGAGCAAGGAATTTTCGGCAATGGGCGcAAGTGACCGAGCAACGCGCGTGCGGGATGACGGCCTT CGGGTTGTAAACCGCTTTTcGGGGGGACGACCCTGACGGTACCCCCGGAACAAGcCCCGGCTAAcTTGTGCC AGcAGcCGCGGTAAGACAGAGGGGGCGAGCGTTGTCCgGAGTCACTGGGCGTAAAGcGCGCGCAGGCG
Sample 82, Forward.	TAGGGCGAATTGGGCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTT ACGGGAGGCAGCAGCAAGGAATTTTCGGCAATGGGCGCAAGCTgACCGAGCAACGCCGCGTGCGGGATGAC GGCCTTCGGGTTGTAAACCGCTTTTCGGGGGGACGATGATGACGGTACCCCCGGAATCAGCCCCGGCTAACT cTGTGCCAGCAGCCGCGGTAAGACAGAGGGGGCGAGCGTTGTCCGGAGTCACTGGGCGTAAAGCGCGCGCA GGCGGCAACCTTAGTGTCGTGTGAAAGCCCCCgGCTCAACCGGGGGAGGCCATGGCAAACTGGGTCGCTCGAG CTGCGGAGAGGCCCCTCGAATTGCCGGTGTAGCGGTGAAATGCGTAGAGATCGGCAGGAAGACCAAGGGGGA AGCCAGGGGGCTGCCGCAGTGACGTGAGGCGCGACAGGTGGGGGAGCAAACCGGATTAGATACCCGGGTAGT CCACGcCGTAAACGaTGACCACTcGGCGTGTGGcGACTATTGACGTCGCGGCGcGCCCTAGCTcAcGcGATAAGT GGTCCgCCTGGGAACTACGAGCGCAAGTTTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGCGGaGT tGTGGTTTAATTtGACGAACCCGcAGAACCTTACCCAGACTGGACATGACGGTGCAGACGGCGGAAACGTCGT CGCTGCGAGGGTCCGTtACAGGTGCTGCATGGCTgTTGTCAGCTtGTGTGGTGAGATGTTgGGTTAAGTcCGAAC GAGGGAACCccTtGG

DNA Sequencing BLAST Results

Sample 81
Accession 1: AF421725.1	Uncultured Chloroflexaceae bacterium	Yellowstone National Park	Boomer, Lodge, Dutton, Pierson
Accession 2: AF445666.1	Uncultured Eubacterium	Yellowstone National Park (Mammoth Hot Springs)	Bonheyo, Fouke, Frias-Lopez, Sanzenbacher
Accession 3: AB041226.1	Roseiflexus castenholzii	Japanese Hot Spring	Hanada, Takaichi, Matsuura, Nakamura
Accession 4: AJ421669.	Uncultured bacterium	Yellowstone National Park (Mushroom Spring)	Nubel, Bateson, Vandieken, Wieland, Kuhl, Ward

Sample 82
Accession 1: AF445666.1	Uncultured Eubacterium	Yellowstone National Park (Mammoth Hot Springs)	Bonheyo, Fouke, Frias-Lopez, Sanzenbacher
Accession 2: AF421725.1	Uncultured Chloroflexaceae bacterium	Yellowstone National Park	Boomer, Lodge, Dutton, Pierson
Accession 3: AJ421649.1	Uncultured bacterium	Yellowstone National Park (Mushroom Spring)	Nubel, Bateson, Vandieken, Wieland, Kuhl, Ward
Accession 4: AB041226.1	Roseiflexus castenholzii	Japanese Hot Spring	Hanada, Takaichi, Matsuura, Nakamura

Discussion

DNA sequencing relies on the inability of polymerases to elongate a nucleotide chain in the absence of a three prime hydroxyl group. Each of the samples were polymerized in the presence of all four deoxynucleotides as well as “stop” dideoxynucleotides. During polymerization the polymerase randomly incorporates the dideoxynucleotide into the chain, which stops elongation of the chain at that point. The polyacrylamide gel used to separate the DNA fragments can separate fragments that differ by a single nucleotide in length. The resulting gel pattern yields the sequence of the DNA (with the assistance of Li-Cor software that reduces the tedious nature of the task). Of course, to start replication polymerase requires a hydroxyl group which is provided by a man made primer so it is not possible to sequence a DNA fragment unless it has been cloned and enough information is known to design the primer.

The sequence analyses of clone numbers 81 and 82 indicate that the bacteria are closely related to each other. This is evidenced by the appearance of many of the same accession numbers from the BLAST analysis. As should be expected, all bacteria from the BLAST hits are phototsynthetic and found in hot springs, mostly from Yellowstone National Park. The similar sequences and therefore close phylogenetic relationship between these bacteria support the conclusions made from the probe hybridization done during week 3 that indicated that both bacteria were red. If the same RFLP analysis had been done on both samples during week 4 we would expect that they would have very similar banding patterns as well. (Due to the number of people in the class I analyzed only #82 with RFLP).

In the future these sequences may be useful for determining the phylogenetic history of the Yellowstone hot spring bacteria. The BLAST hits also indicate that the bacteria are related to Roseiflexus bacteria and to bacteria found in Japanese hot springs. Previous research has also indicated this relationship as well, and further study of these two types of bacteria may prove useful in the development of the phylogeny of the Yellowstone bacteria.