Biology 475

Molecular Biology

Lab Five - DNA Sequence Analysis

Elizabeth Jacobsmuhlen

Spring 2003

Introduction

To use information already known about our DNA samples and perform DNA sequence analysis on it. This is a more specific (single nucleotide bands) and targeted (primer used) way to gain more information about our DNA that was cloned in lab four.

	DNA Sequencing Methods
Reaction Set-up Methods: The picture to the right shows the mixing of our sample DNA (clone #73 and #74) with taq pol, buffer, all four dNTPs, and the proper dd (ddATP, ddGTP, ddTTP, ddCTP), and cocktail made of buffer, primer, and polymerase. Each clone number in a separate four well strip.
Gel Pouring Methods: The picture to the right shows the insertion of the polyacrylamide solution in between the two clean glass plates.

DNA Sequencing Results

Sample 73, Forward

>Liz Sample #73

tRGtAAtAYGMTcaCTATAGGGcGAATTGGGCcTCTAGATGATGctcGAgcGGCGCAGTG

TGATGGATATCTGCAGAATTCGCCTTACGGGAGGCAGCAGCAAGGAATTTTCGGCAATGG

GCGCAAGcTGACCGAGCAACGCCGCGTGCGGGATGACGGCCTTCGGGTTGTAAACCGCTT

TTCGGGGGGACGACCCTGACGGTACCCCCGGAACAAGCCCCGGCTAACTCTGTGCCAGCA

GCCGCGGTAAGACAGAGGGGGCGAGCGTTGTCCcGAGTCACTgGGCGTAAAGCGCGCGCA

GGCGGCAACCTAAGTgTCGCGTGAAAGCCCCCGCTCAACCCGGGGAGGCCATgGCAAACT

gGGTCGCTCgAGCTCGGAGAGGCCCCTCGAATTgCCcGTGTACGTGAAATCGTAAGATCC

cCAaAAgACCAAGGGGAACCAGGGGCTtCCCCA

Sample 74, Forward

>Liz Sample #74

AATTGTAATACGACTCACTATAGGGCGAATTGGGCCCTCTAGATGCATGCTCGAGCGGCC

GCCAGTGTGATGGATATCTGCAGAATTCGCCCTTACGCGGTTACTAGCAACTCCGGCTTC

ATGCAGGCGGGTTGCAGCCTGCAATCCGAACTACGaCCGGCTTTGGTGGATTGGCTCCCC

CTCGCGGGTTGGCTACCCTCTGTACCgGCCATTGTAGCGTGTGTGTAGCCCTGGACATCA

AGGCCATGCTGACTTGACGTCATCCTCACCTTCCTCCCGCTTTCAACGGGCAGTCCCGCC

AGACACCTGTAACTGACGGCGAGGGTTGCGCTCGTTACCgGACTTAACCGAACATCTCAC

GACACGaGCTGACGACAGCCATGCAGCACCTGTGGCGGCTCCCGAAGGTCGCTCCCCTTT

CAGGTCGCTACCACCGCCATGTCAAGCCCAGGTAAGGTTCTTCGTGTAGCCTCGAATTAA

ACCACACGCTCCGCTGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAACCTTGCGG

CCGTACTCCCCAGGtGGCGGACTTATCACGTTCGCTTCGGCACGGCAGCTTCCACCgCCA

CACCTAGTCCGCATCGTTTACAGCATGGACTACCGgGGGTTTCTAATCCCGTTCGTCCCC

ATGCTTTCGCGCCTCAGGTCAGGTCAGGCCCAGCGCGCCGCCTTCGCCACTGGTGTTCCT

CCGGATCTCTACGATTTCACCACTACACCCGGAATTCCACGcgCCTCTACCTGCCTCTAG

cTCAGcAGTCTCGCATGTCCtgCCCCGTTGAGCCtgAGcgCTTTAATAGgGTTGTTAGCCGCgG

Top Four Different Blast Species for Each Sample Clone

Sample 73

Accession Number	Name/ Phylum	Source	Reference
AF421725	Uncultured Chloroflexaceae bacterium/ bacteria	Hot spring in Yellowstone National Park	Appl. Environ. Microbiol, Boomber, S. M., Lodge, D.P, Dutton, B. E. and Pierson, B; PUBMED 11772644
AF445666	Uncultured eubacterium/ Bacteria	Mammoth Hot Springs, Yellowstone National Park	Bonheyo,G.T., Fouke,B.W., Frias-Lopez,J. and Sanzenbacher,B.; Unpublished
AB041226	Roseiflexus castenholzii/ Bacteria	Japanese hot spring	Appl. Environ. Microbiol, Boomber, S. M., Lodge, D.P, Dutton, B. E. and Pierson, B.
AJ421665	uncultured bacterium/ Bacteria	Hot spring microbial mats	Appl. Environ. Microbiol; Nubel,U., Bateson,M.M., Vandieken,V., Wieland,A., Kuhl,M. and Ward,D.M.; Medline 22188273

Sample 74

Accession Number	Name/ Phylum	Source	Reference
AJ519644	Uncultured Chlorobi bacterium/ bacteria	Uranium mining waste piles and mill tailings	Selenska-Pobell S.I.
AJ306745	Uncultured Bacterium/ bacteria	Environmental samples from scientists in Germany	Schloetelburg,C.
AF027036	unidentified green non-sulfur bacterium OPB65/ Bacteria	Yellowstone hot spring	Medline # 98101476 J. Bacteriol
AF018195	uncultured eubacterium env.OPS 12/ Bacteria	thermal gradient in a stream near Obsidian Pool, Yellowstone National Park	Graber,J.R., Kirshtein,J.D., Beveridge,T. and Reysenbach,A.-L.; Biochemistry and Microbiology

Discussion

In this lab the DNA template population and the information known about it was used to determine a proper primer to be made/bought in order to for sequencing to be performed. The sequence was obtained from the ÒcloneÓ of a gene. The primer must then stick to the DNA once it is denatured with heat and in single-stranded configuration. DNA Pol will build a complementary DNA strand when a template, correct bound primer, and nucleotides are available. The nucleotides were added to the mixture (dNTPs) and ddNTPs were also added (ddGTP, ddTTP, ddCTP, ddATP) into the proper sample tube. The ddNTPs (di-deoxy-nucleotide triphosphate) lack an OH group and cause a termination of growth of the complimentary chain allowing a single shot to be made of every monmer (nucleotide) being added. This cocktail is then loaded onto a gel and sequenced through use of the Li-Cor technology/program.

As discussed in week #4 lab, based on the effectiveness of the enzymes used it was supporting the two samples are related, but are not the same type of bacteria. Theoretically if the Clone #73 and #74 were the same type of bacteria then the each type of restriction enzyme would have been equally effective on both types of clones. In theory, if the two samples were the same type of bacteria with RFLP performed on them would have the same first two hits in BLAST. As it can be seen from the chart above the #1 blast hits for clone #73 and #74 were different. Indicating that the sequence obtained from our samples clone #73 and #74 was not an exact match to the same type of bacteria. In fact two out of the top four different organism matches were the same and two were different. This information is in agreement with the information obtained from the enzymes used in lab four. One of the enzymes worked well for both clones, one enzyme worked well for one clone but only moderately well for the other, and one enzyme was not effective for either clone. This information from RFLP suggests that the restriction enzyme cut at different positions or cut at the same position either of which resulted in a different sequence.

Week #3 lab results showed that clone #73 reacted with a positive result to red bacteria. The green and all bacteria probes were inconclusive for #73 and #74. Red bacteria probe appeared to be negative for clone #74. Due to the blots appearance they were very difficult to read and were in general inconclusive. However, when lab three is taken into consideration along with the library screening and RFLP data all suggest that clone #73 is a type of red bacteria and #74 is a type of green bacteria.