Geochemistry & Ecology of Red Mat Systems (GERMS)

Undergraduate Summer Research Program

 

Week Five

DNA Sequence Analysis

 

Sequencing Reaction Set – Up

 

Procedure:

Tubes for Imperial. We loaded the reactions with stop G, stop A, stop C, and stop T to our samples of DNA.

Procedure:

Heating up the DNA to denature it, using the aid of the PCR machine.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Preparing Gels:

 

Procedure:

Jen loading the Gel plate into the DNA sequencing machine.

Procedure:

Capping the electrode and then hook up the electric current.

 

Reaction Finish and Load:

 

Procedure: Maria is loading her samples onto the DNA sequencing machine. It uses a laser light to scan back and forth across the samples. The result is a gel that shows the differences in A,G,T,C.

 

Reading, Editing and BLASTing Sequences

 

SEQUENCE INFORMATION

 

Clone Number Analyzed: Imperial Green 1-5

 

Edited Sequence:

 

Imperial 1:

 
GAAACGTAaGaGtctCaacaagggggggcgaacgggGCCCcCTagAAGCAGcgTCGAGCGGcCGaCgAgtGTGA
GGATATCTGCAGAATCGgCCttgAGCTCGaGCgcCCgCCCcgCGCTTACCTTGTTTACGgACTTcACCCCAGGT
CATCAGCCCTGCCTTAGGTGCCCCcCTCCcgCGaACGgTTAGGgAACCAACTTCGGGCcGTGGCCAACCTTCCC
cATCgggTgACGgGCGGTGTGTACAAggcCCGgGAACGTAtTTCACCGCCGTATGCTGACCgGCGaTTACTAGC
GatTaCGcCTTCATGcAGGcGaGTTgtAGCCTGcAATCTGaaCTGAGCCTtGGtTTACGGGATtgaGCGcACCC
TCGcGGGctTgGCAAcCCcCTgTgtTtCccAGCAgTTgTAGTACGTGttGTAGCCCAGGGaCGtAAGGGgGCAT
ttTTGaCTTggACGTCA

Imperial 2:

 
CTgTAGAtGcATgctcaAGcgacCagcACAgacGccgAcCCcCACActTGTAgtTTttCgAcCccacgccaagG
cGttCtTtttcCccCaacAatAtatACGtcTcTaTccCTCTCagACCagagacaCAaacCtCtgTCAGcAAGGt
cGaaTTcCAGcACACTTGAgcGGcCgcTTAcTAGTGGaTCCgaGcTtGGTaccacGtTTgGgGTccTtcTggTT
cTcGtTgTTTtcTgTgTGcccTTgTTcTgcGgTtcgccTTtaccccccccTccGcggcGgccGtcTcccGTGTa
cacGcTGGGGTGGcTactGcGTGcGgTccgTtcccTTccTTGtGtTgGGGTTcgTgccccGgT

Imperial 3:

 
ttctgaaTTgTAaTacGccTcacTaTaGGgcGaattgGGCCCTCTAGATGCATGCTCGAGCGGCCGcCAgTGTt
GAtggatAtctgcAggattcggtcttaTgtggTcgagtgggCGtccggggTTaCCTTGTTACGactgaGtcCCa
atcAGGGTGTcCatCtctCGgCGCACCGGGGTGCGACTTCGGaTGTcCTcCCCTTTcGTGGCTTGACGGGCGGT
GTGTACAAGGCTCAGGaACACATTcCCCGCGTTGTTGcTGaCACGgGAGTACTAGCGATTCCgaCTTCATGAAG
GCGAGTTGCAGCCTTCAATCTGAACTGGGGTGTGTTTTTTtGGCTTtGcTCCgACTCGgGTCTTCGCTTCCCtT
TGtTCACACCATTgTAGCACGtGTGCAGCCCTAGACGTAAGGGCCATgaTGaCTTgACGTcGTCCCCACCTTcC
TcGCgGTTTCACAcCGGCGGTCCTGTTAGaGTGCCCTcGGGtTGcAACTAACAGCAGGGGTTTcGTCGcTATGG
GACTTAACCCGaCAtgTgAGacACGaGcTGaCGaCAGCCATGCAgCAcCTGTGCaGCGttCCtaG

Imperial 4:

 
CgcgccTtttCctcaGcATaCaTcTCGGgaGccttgGGAAATATCGCtGACTGATAGCGAGGAAGAACGTGTGc
TggTtTcTtTTtTtCgtgGggtaggaAATTgGATAGCGAgtaAGtAagTgTGTTTCAATtGttCAGcaTttGga
aaaCCtactcgCaACactagttttTGCAATgaaTaAATTGaGGGTTCGATACAGAACATTATCGTGACGAATCA
TcTAGttCCcTatGCAGggAGagTGTGTCACCGcAAAgggcCAGTcTTaCAAgaCGACTGAAGCTATgAGCTTc
ATcGAGCTTaACGATTaCTGaCGTaGCGCTTtACGCATtACCTaAgCcATtCGGcGCCCTttCTgCgGtTaGcT
aAAAATaGtGgGTTTtACTATATTaTGTtGTTAACAAcTTGTCGACAGTGTGTCGAAAttGcGGAAGTCCGgGA
ACTgTggGcAatTGCCAGTTCTAAAACaATTAgATATgCcTTaCctaCTACGAgGTtATTCAAccCCGTC

Imperial 5:

 
CCcTCTaGATgcAtAtCGaGcGGgCGCAGtGTGaTGGATATcTCCagatTCgACC

 

BLAST HITS

Be sure to use different hits (uncultured = same;  go down until you find specific species, including cultured isolates)

 

For source, I am looking for where it came from (e.g. termite gut);  if none is listed, read the title reference and abstract (if published) to glean this information.

Top Accession #

Organism Name

Source

X03538 K01982 X01296

Synechococcus sp. PCC 6301

Unknown

AF239693

Gemmata-like str. CJuql4

Freshwater and soil

 

 

Discussion:

This is the final week, and the research project is coming to a close for us students. I have to say that I thoroughly enjoyed doing this research and would like to do it again next year if possible. This week we did DNA sequencing, which is a very thin gel .2mm. It took awhile to get used to loading the gels. We then received our data back from our samples and then ran these images on the computer to sequence the DNA. From this sequencing we ran our data online at BLAST and then matched our DNA sequence with the database. I saw much research ongoing about cultured bacteria. It was a very useful database that gave the names of millions of bacteria and other things, such as mRNA of a silk worm. You simply sequence the DNA of the organism you are working on and match it up against the database of BLAST. You can then see how closely matched your sample is to the top accession number and then determine how accurate your sequencing was, and how closely related your organism is with the documented organism.  Imperial sample #1 came out to match that of Synechococcus sp. PCC 6301 most closely. Imperial sample #3 came out to match that of Gemmata-like str. CJuql4 the most closely.