Biology 475

Molecular Biology

Lab Five and Six – DNA Sequence Analysis

Kelly L. Shipley

Copyright 2003

 

Introduction

 

Using ddNTPs we will put our samples through PCR to amplify replication getting a map of the DNA.  As each nucleotide is added to the strand during replication, replication is stopped by the termination mix.  This will give us a reading where each strand will have only a one nucleotide difference.  To see these differences and be able to map out the sequence, we will use gel electrophoresis with stronger resolution than we have used before, fluorescent tags and a sophisticated sequencer.  The sequencer will read and display, on the computer, the size and nucleotide identity of each band, and using specific software tools we will be able to read the sequence of DNA.

 

 

 

 

 

MY FRIEND LIZ LOADING A GEL!

 

 

 

DNA Sequencing Methods

Reaction Set-Up Methods:  PCR tubes lined up after receiving cocktail solution.  The four rows indicate (from top to bottom) Sample #52 Forward, Sample #52 Reverse, Sample #53 Forward, Sample #53 Reverse.  The four tubes in each row represent different Termination Mixes, from left to right, Stop T, Stop G, Stop A, Stop C.  After adding all ingredients to the cocktail, they are added to the PCR machine for cycling.

 

 

Gel Pouring Methods:  After the acrylamide gel mix is added in between the two plates, the lane comb is placed upside-down to create an area for loading the gels with our samples.  The comb press is laid on top of the comb to secure it in place while the gel polymerizes.

 

 

 

 

 

DNA Sequencing Results

Sample 52, Forward

AATACGACTCACTATAGGGCGAATTGGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGT

GTGATGGATATCTgCAGAATTCGCCCTTACGCGGTTACTAGCAACTCCGGCTTCATGCAG

GCGGGTTGCAGCCTGCAATCCGAACTACGACCGGCTTTGGTGGATTGGCTCCCCCTCGCG

GGTTGGCTACCCTCTGTACCGGCCATTGTAGCGTGTGTGTAGCCCTGGACATCAAGGCCA

TGCTGACTTGACGTCATCCTCACCTTCCTCCCGCTTTCAACGGGCAGTCCCGCCAGACAC

CTGTAACTGACGGCGAGGGTTGCGCTCGTTACCGGACTTAACCGAACATCTCACGACACG

AGCTGACGACAGCCATGCAGCACCTGTGGCGGCTCCCGAAGTCGCTCCCCTTTCAAGAAG

CTACCACCGCCATGTCAAGCCCAGGTAAGGTTCTTCGTGTAGCCTCGAATTAAACCACAC

GCTCCGCTGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAACCTTGCGGCCGTACT

CCCCAGGTGGCGGACTTATCACGTTCGCTTCGGCACGGCAGCTTCCACCGCCACACCTAG

TCCGCATCGTTTACAGCATGGACTACCGGGGTTTCTAATCCCGTTCGCTCCCCATGCTTT

CGGCCTCAGCGTCAGGTCAGGCCCAGCGCGCCGCCTTCGCCACTGGTGTTCCTCCGGATC

TCTACGcATTTCACCACTAcACCCgGAATTCCAcGcGCCTTTACCTGCCTTTAGTcAGAG

TTTCGaATgTCcTgg

Sample 52, Reverse

CATGATTACGCCAAGCTTGGTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGCT

GGAATTCGCCCTTACGGGAGGCAGCAGCAAGGGATATTGCGCCAATGGGCGAAAGCCTGA

CGCAGCAACGCCGCGTGCGGGAAGAAGGCCTTCGGGTTGTAAACCGCTTTGAGAGGGGAC

GAGGCAGGACGGTACCCTCAGAACAAGTCTCGGCTAACTACGTGCCAGCAGCCGCGGTAA

AACGTAGGAGGCGAGCGTTATCCgGAGTTACTGGGTGTAAAGGGCGTGCAGGCGGCTGGG

CAAGACGTATGTGAAAGCGCTCAGGCTCAACCGGGCGAGGACATGCGAGACTGCTGAGCT

AGAGGCAGGTAGAGGCGCGTGGAATTCCgGGTGTAGTGGTGAAATGCGTAGAGATCCcGA

GGAACACCAGTGGCG

Sample 53,

Reverse

ATGATTAGGGAAGCTTGGTACCGAGCTCGGATTCACTAGTAAAGGGtGGtAGTGTGGTGG

AATTtGCTTACGGGAGGAAGAAGAAAGGAATATTGATGAATGGGGGAAAGATGaTGGAAG

TGAaGCTGagGtGGggGATGTAAGGGGTTTGGGTTGTAAAGgCgTTTTTTGgGGGAtgTa

GtAAGGACGgTAgTAGAGGCATAAGAGGCGGGGAAATAAGTGgaAGGAGaGtGTgTAAAA

aGTAGTgGaGtGAGGTTATTGGATTagTGAGGtGTAAAGGggTTGAGGAGgTtcGTAAGT

TGGGGGTGAAAGACCGGGTTAAAcGGGGgAgTgAGtaAAATAGTCGGGGGATTGAGGGAA

GAAGAGGGAGGTGGAATTCAGGGTGTAGTGGTGAAATGtGTAGATATTGGGAGGaACAAG

CGTGGGGAAGGtGGTTTGTTCGGgTtTTTCAAGgTGAAGGGAAAgGAGGGGaGGAACggA

TTAGATATTTGGtAGTaTGGTGTAAAGTTCATGTGGTTGGaGaaTTTTTTTGtAGAACTT

AGCTTAAATCCGgTGGaATAGCCAaTAAATGAAGATTGGGGgcgAA

Sample 53, Forward

AGaTaaTATAGGGGATTGGGCcTcTAGATGATGTTGAGGGCGCAGTGTGATGGATATcTG

AGAATTCGCCTTAcGGGGTTAaTAGCAAcTCCAGaTTCATGCAGGCGGGTTGCAGcCTGC

AATcCGAACTGAGACCGGGTTTGGGGGATTGGCTcCGCTCACGGTTAGgAACCCATTGTC

CCGGCCATTGTAGCGTGTGTGTAGCCCTGGgcATAAAGGCCATGCTGACTTGacGTCATC

CCCACccTTCCTcCAGTTGTCCCCGGGcAGTCCCCCTAGACACATGTAACTAgTGGGCAG

GGGTTGTGCTcGTTCCGgGACTTaACCCGACACCTCACGGcacGAGCTGACGACAGcCAT

GCAGCACCTGTGCAGGCTCCCTTgCgGGTCGGTCACCTTTCGGctCCCTAcCACCTGCAT

GTCAAGCCCAGGTAAGGTTCTTCGTGTAGCATCGCaTTAaACCACACGCTCCGCTGCTTG

TGCGGGCCCCGCCAATTCGTTTGAGTTTTaGCCTTGCGGcCgTAGTCCCCAGGCGGGATG

CTTAACGcGTGaGCTTTCGgCAgGGAAGGTAGATcCCTCCCaCACCCaGCAtCCCaCGTT

TACGcCcAGGAT

 

DNA Sequencing Results

Top FOUR DIFFERENT BLAST species for each

Sample # 52 Forward

Name/Phylum

Source

Reference

AJ519644

 
Phylum: Bacteria
Name: uncultured Chlorobi bacterium
 
Bacterial communities within uranium mining waste piles and mill tailings in Germany

 

Unpublished

Author: Geissler, A. et al.

AJ306745

 
Phylum: Bacteria
Name: uncultured bacterium

 

No information

 

(Germany)

Online Journal Publication

Author: Schloetelburg, C.

AF050566

 
Phylum: Bacteria
Name: Chloroflexi; uncultured eubacterium WCHB1-62

 

microbial communities associated with an aquifer contaminated with hydrocarbons (mainly jet fuel) and chlorinated solvents

Medline: 98432811

AF027043

 
Phylum: Bacteria
Name: Chloroflexi; unidentified green non-sulfur bacterium

 

Obsidian Pool (OP), a Yellowstone National Park hot spring

Medline: 98101476

 

Sample # 52

Reverse

Name/Phylum

Source

Reference

AJ519644

 
Phylum: Bacteria

Name: uncultured Chlorobi bacterium

 
Bacterial communities within uranium mining waste piles and mill tailings in Germany

 

Unpublished

 

Author:  Geissler, A. et al

AF323744

Phylum: Bacteria

Name: uncultured bacterium

 
bacteria members associated with benzoate degradation in the methanogenic consortium

 

Unpublished

 

Author: Wu, J.H. et al

AF027044

Phylum: Bacteria

Name: Chloroflexi; unidentified green non-sulfur bacterium

 Obsidian Pool, a Yellowstone National Park hot spring

Medline: 98101476

AJ518365

Phylum: Bacteria

Name: unidentified bacterium

 
microbial communities in sediments of water

 

Unpublished

 

Author: Bleul, C.

 

Sample # 53

Reverse

Name/Phylum

Source

Reference

AF027035

Phylum: Bacteria

Name: unidentified green non-sulfur bacterium

Obsidian Pool, a Yellowstone National Park hot spring

Medline: 98101476

AF419669

Phylum: Bacteria

Name: uncultured bacterium

Microbial communities in hydrothermally active sediments of the Guaymas Basin

Medline: 21914114

AF424402

Phylum: Bacteria

Name: uncultured Chloroflexi bacterium

surficial sediment core obtained from an Antarctic continental shelf area

Medline: 22617432

AF524862

Phylum: Bacteria

Name: bacterium K-5b9

Acidophilic Methanogenic Community from Sphagnum Peat Bog

Unpublished

 

Author: Sizova, M.V. et al

 

Sample # 53

Forward

Name/Phylum

Source

Reference

AF027035

Phylum: Bacteria

Name: unidentified green non-sulfur bacterium

Obsidian Pool, a Yellowstone National Park hot spring

Medline: 98101476

AF419665

Phylum: Bacteria

Name: uncultured bacterium

Microbial communities in hydrothermally active sediments of the Guaymas Basin

Medline: 21914114

AF424395

Phylum: Bacteria

Name: uncultured Chloroflexi bacterium

surficial sediment core obtained from an Antarctic continental shelf area

Medline: 22617432

AF423186

Phylum: Bacteria

Name:  toluene-degrading methanogenic consortium bacterium

toluene-degrading methanogenic consortium enriched from creosote-contaminated aquifer material

Medline: 20050001

 

Discussion

 

            DNA sequencing is a very specific process whereby we use ddNTPs and dNTPs and primers labeled fluorescently to allow for replication of our target sequence, and subsequent stopping of the sequence at each specific nucleotide.  The sequencing machine then has the capability to read the gel one nucleotide difference at a time, giving us a map to be able to read and sequence.  This procedure was followed by work on the computer with a program designed to read the sequencer output, BLASTing our sequence using NCBI and determining at that point the similarity between our sequences and known sequences in the GenBank database. 

            First, sample #52 was sequence using both the forward and reverse primer.  In doing this, we are able to see both the sequences and use the BLAST tool to analyze their similarities to other organisms.  #52 was found to be mostly related to uncultured Chlorobi bacterium, which is a form of green sulfur bacteria.  This similarity was found for both the forward and reverse sequences for sample #52 but this does not correlate to the results from our slot blotting which show negative results for sample #52 when testing for the presence of 16s rRNA sequences specific to green bacteria.  The fact that it related to a sulfur bacterium relates to our sample’s origin in the microbial mats of Yellowstone.

            Second, sample #53 was also sequenced using the forward and reverse primers.  When using the BLAST tool to determine similarity to other sequenced organisms, sample #53 was found to be most closely related to unidentified green non-sulfur bacterium.  Again, this does not correlate to our slot-blotting results which did not show positive results when using the green bacteria specific probe.  Another common hit when BLASTing sample #53 was Chloroflexi bacterium, again a type of green bacteria. 

            These results do not correlate to the results from the slot blotting tests we performed earlier in the term.  They also do not correlate to the gel electrophoresis results we obtained after our first restriction digest with EcoRI which showed amazing correlation between all of the insert identifications.  These were assumed to be 16s rRNA from red bacteria based on recognition of different 16s rRNA bands on other student gels specific to green bacteria.  I felt this was later confirmed by the slot blotting positive results with the 16s rRNA red-specific probe.