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Advanced Environmental Biotechnology II

Lecture 5 - Principles and applications of DNA

sequencing

Look here for a good animation of the process.

http://www.dnalc.org/resources/animations/cycseq.html

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DNA Sequencing

DNA sequencing is used to find out the order of nucleotides in a sample of DNA A major goal of molecular genetics is to find the sequence of a gene with its function. The most popular method for doing this is called the dideoxy method.

4Image by LadyofHats Mariana Ruiz

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Dideoxy Method

DNA is made from four deoxynucleotide triphosphates.

Each new nucleotide is added to the 3′ -OH group of the last nucleotide added.

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The nucleosides and their mono-, di-, and triphosphates

Base Nucleoside Nucleotides

DNA

Adenine (A) Deoxyadenosine dAMP dADP dATP

Guanine (G) Deoxyguanosine dGMP dGDP dGTP

Cytosine (C) Deoxycytidine dCMP dCDP dCTP

Thymine (T) Deoxythymidine dTMP dTDP dTTP

RNA

Adenine (A) Adenosine AMP ADP ATP

Guanine (G) Guanosine GMP GDP GTP

Cytosine (C) Cytidine CMP CDP CTP

Uracil (U) Uridine UMP UDP UTP

7From http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/N/Nucleotides.html#nucleosides

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Pasted from <http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/ddTTP.gif>

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1 - Denature the DNADNA dissolved in water is heated until the two strands of DNA come apartThe temperature when this happens is called the Tm If we cool the tube again the two strands of DNA reform double-stranded DNA This is called 'annealing' or 'hybridization'Only complementary strands will come together

10http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html

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2. Add a Primer

For each strand, we provide a primer, which is a short piece of DNA that sticks to one end of the strand

Cool the mixture

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3. Add Precursors and Enzymes

The mix includes the template DNA, free nucleotides, a DNA polymerase enzyme and a 'primer'

The primer sticks to its target and DNA polymerase starts growing the primer

Normally a new strand of DNA results

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Use a Different Base

What happens if I add this instead?

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Dideoxyribonucleotide can be used just like a normal deoxyribonucleotide

But it has no 3' hydroxyl group

When it's added to the end of a DNA strand, the DNA strand won’t grow

Just a fraction of the nucleotides are dideoxy nucleotides

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If Dideoxynucleotide is used

Replicating a DNA strand in the presence of dideoxy-T

Most of the time when a 'T' is required to make the new strand, the enzyme will get a good one

5% of the time, the enzyme will get a dideoxy-T, and that strand stops growing

17Diagram from http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html

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Sooner or later all of the copies will be stopped by a dideoxy T

There will be strands stopping at every possible T along the way

All we have to do is find out the sizes of all the strands

19Diagram from http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html

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Gel electrophoresis can be used to separate the fragments by size and measure them.

21Picture by Jeffrey M. Vinocur

Gel electrophoresis

machine

These leads make an electric

difference between the ends of

the gel

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Drawn by Magnus Manske

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Find the Fragment Size

Here are the results of a sequencing reaction run in the presence of dideoxy-Cytidine (ddC)

24Diagram from http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html

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Fluorescent Dideoxynucleotides

We need to be able to see the fragmentsDideoxy nucleotides can be changed to glow under UV lightThe dideoxy-C, for example, glows blue Now put the reaction products onto an electrophoresis gel Smallest fragments move fastest The positions and spacing shows the relative sizes

26www.kensbiorefs.com/MolecularGen.html

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Four Fluorescent ddNucleotides

Run the reaction with all four of the dideoxy nucleotides (A, G, C and T) present, and with different fluorescent colors on each

The gel will tell the sequence of the DNA

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29Diagram from http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html

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Automated sequencing Gel

Run DNA replication reactions in the presence of small amounts of all four of the dideoxy terminator nucleotides

The gel separates the resulting fragments by size and we can 'read' the sequence from it, bottom to top

A machine can run the gels and to monitor the different colors as they come off the bottom

This is an automated DNA sequencer.

31Diagram from http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html

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Automatic Sequencer

An ultraviolet laser shines through the gel near the bottom and scans side to side

Bands of fluorescent colors pass through its beam

As many as 96 'lanes' of samples run in one gel

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Automating Sanger Sequencing

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Reading the Sequence

The computer reads the sequence from the gel

It draws a graph of the colors found in one 'lane' of a gel.

The computer prints the nucleotide sequence across the top.

35Diagram from http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html

36http://www.cityofhope.org/NR/rdonlyres/E86F0DFD-3A15-4E16-8C0E-BAA4D20CDA24/0/HomePageImage.jpg

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Sequencing a Genome

We can get the sequence of a fragment of DNA as long as 700 nucleotides

If we need to read longer sequences we can break them up into smaller pieces

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Some Applications

Look at the following titles of scientific papers, and their abstracts.

How do you think that DNA sequencing would have been used in these?

Include what reason it was used.

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Influence of repeated prescribed burning on the soil fungal community in an eastern Australian wet sclerophyll forest 

Soil Biology and Biochemistry, In Press, Corrected Proof, Available online 28 July 2006, Brigitte A. Bastias, Zhiqun Q. Huang, Tim Blumfield, Zhihong Xu and John W.G. Cairney

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A long-term prescribed burning experiment, incorporating replicated plots that receive burning biennially (2 yr burn) or quadrennially (4 yr burn) and unburned controls, has been maintained in a wet sclerophyll forest at Peachester, Queensland, Australia since 1972. In 2003 we extracted DNA from soil collected from the experimental plots and investigated the influence of the burning on the soil fungal community by comparing denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified partial rDNA internal transcribed spacer regions (ITS1). Canonical analysis of principal coordinates (CAP) of the DGGE profiles of the upper 10 cm of the soil profile grouped the data strongly according to treatment, indicating that both burning regimes significantly altered fungal community structure compared to the unburned controls. In contrast, no obvious trend was observed for soil from a depth of 10–20 cm of the profile. Sequencing of selected DGGE bands found no obvious patterns of presence/absence of taxonomic groups between the treatments. Analysis of soil nitrogen and carbon by mass spectrometry indicated that total soil C and N, along with both gross and net N mineralisation, were significantly lower in 2 yr plots compared to control and 4 yr plots.

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Methanogenic diversity in anaerobic bioreactors under extremely high ammonia levels 

Enzyme and Microbial Technology, Volume 37, Issue 4, 1 September 2005, Pages 448-455 Baris Calli, Bulent Mertoglu, Bulent Inanc and Orhan Yenigun

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To evaluate the effects of high free ammonia nitrogen on methanogens, five laboratory scale UASB reactors seeded with different sludges were operated for 450 days. Throughout the experimental period, free ammonia concentration was gradually elevated up to 750 mg/l. Changes in methanogenic population were investigated by using 16S rDNA/rRNA based molecular methods such as denaturing gradient gel electrophoresis (DGGE), fluorescent in situ hybridization (FISH), cloning and DNA sequencing. Generally, in all of the reactors, moderately high COD removal was achieved in the range of 77–96%. However, in three of the reactors, propionate degradation and in one of them, acetate removal was influenced more severely. In no way, neither of the phases was inhibited in reactor 4 (R4), which was seeded with a biomass concentrated from a landfill leachate. On the other hand, as free ammonia level elevated, instead of a community shift, single coccus shaped Methanosarcina cells previously predominant at low free ammonia concentrations turned into stringent multicellular units, in all of the reactors.

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The impact of sludge amendment on methanogen community structure in an upland soil 

Applied Soil Ecology, Volume 28, Issue 2, February 2005, Pages 147-162 S.K. Sheppard, A.J. McCarthy, J.P. Loughnane, N.D. Gray, I.M. Head and D. Lloyd

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In the EU, municipal sewage sludge application to agricultural land has increased dramatically since the ban on dumping at sea came into effect in 1998. There are many concerns related to potential contamination and reduction in plant productivity. In this study, the aim was to assess the impact of repeated long-term soil amendment with anaerobically digested sewage sludge on methanogen diversity in an upland soil ecosystem. Sludge-treated and untreated upland soil samples as well as samples of the sludge used, were analysed for the diversity of methanogens using TGGE, PCR-RFLP and DNA sequence analysis of approximately 490 bp of the mcrA operon. PCR analysis using mcrA specific oligonucleotide primers confirmed the presence of methanogen DNA in treated and untreated soil samples and in sewage sludge. TGGE was used to describe the diversity of methanogen mcrA sequences and the differences in community structure between samples. Ninety-six mcrA gene PCR products were screened using RFLP analysis representing methanogen DNA amplified from anaerobically digested sewage sludge, control soils and sludge treated soils. Fourteen RFP's were detected in all treatments, five of which were common to all three treatments.

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Thirty-eight cloned amplimers were selected for sequencing and phylogenetic analysis. These included representatives of each RFP. From control soils, sludge and sludged soil samples 15, 16 and 7 clones were sequenced, respectively. Phylogenetic analysis suggested that they represented hitherto uncharacterised mcr genes; 35 of the clones fell into 7 clusters supported by moderate to high bootstrap values. The diversity of methanogens in an upland soil (treated and untreated) and sludge was evaluated and marked differences in the diversity of the methanogen communities was observed between the treatments. Our results indicate that sludge application may reduce soil methanogen community diversity.

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Differentiation and identification of iron-oxidizing acidophilic bacteria using cultivation techniques and amplified ribosomal DNA restriction enzyme analysis 

Journal of Microbiological Methods, Volume 60, Issue 3, March 2005, Pages 299-313 D. Barrie Johnson, Naoko Okibe and Kevin B. Hallberg

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Acidophilic iron-oxidizing microorganisms are important both environmentally and in biotechnological applications. Although, as a group, they are readily detected by their ability to generate ferric iron (resulting in a distinctive color change in liquid media), these microbes highly diverse phylogenetically. Various other characteristics, such as optimum growth temperature, response to organic carbon sources, and cellular morphologies, facilitate, in some cases, identification of isolates to a genus or species level, although this approach has limitations and may give erroneous results. In this study, a combined approach of using physiological traits together with amplified ribosomal DNA restriction enzyme analysis (ARDREA) has been successful in identifying all known acidophilic iron-oxidizing bacteria to the species level. Computer-generated maps were used to identify restriction enzymes that allow the differentiation of the acidophiles, and these were confirmed experimentally using authentic bacterial strains.

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To test further the validity of this approach, six acidophilic moderately thermophilic iron-oxidizing bacteria isolated from Montserrat (West Indies) were analysed using the ARDREA protocol. Three of the isolates were identified as Sulfobacillus acidophilus-like, and one as Sulfobacillus thermosulfidooxidans-like bacteria. The fifth isolate gave DNA digest patterns that were distinct from all known strains of iron-oxidizing acidophiles. Subsequentsequencingof the 16S rRNA genes of these isolates confirmed the identity of the four Sulfobacillus isolates, and also that the fifth isolate was a novel species. Schematic diagrams showing how ARDREA may be used to rapidly identify all known acidophilic iron-oxidizing bacteria are presented.

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Identification of novel eubacteria from spent mushroom compost (SMC) waste by DNA sequence typing: ecological considerations of disposal on agricultural land 

Waste Management, Volume 24, Issue 1, 2004, Pages 81-86 M. Watabe, J. R. Rao, J. Xu, B. C. Millar, R. F. Ward and J. E. Moore

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A small study was undertaken to examine the microbiological characteristics of spent mushroom compost (SMC), which is the major waste by-product of the mushroom industry and which is regularly disposed off by application to agricultural land. The primary aim of this study was to examine SMC for the presence of faecal bacterial pathogens, including Campylobacter spp., Salmonella spp. and Listeria monocytogenes. Secondly it was desirable to quantify bacterial and fungal populations within SMC, and also qualitatively identify the diversity of bacterial populations within SMC, through employment of rDNA PCR and direct sequencing techniques on the culturable microflora. Conventional microbiological analyses of SMC material (n=30) from six commercial operations in both Northern Ireland and the Republic of Ireland, failed to detect Salmonella spp, Listeria spp. or Campylobacter spp. in any of the SMC material examined. Total aerobic plate counts gave a mean count of log10 7.01 colony forming units (cfu) per gram SMC material (range: log10 6.53–7.52 cfu/g). Fungal counts gave a mean count of log10 4.57 cfu per gram SMC material (range: log10 3.93–4.98 cfu/g).

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From a total of greater than 50 colony picks, a total of 12 bacterial morphotypes were identified and were further examined by employment of partial 16S rRNA gene amplification and sequencing techniques, yielding several genera and species, including Bacillus licheniformis, Bacillus subtilis, Klebsiella/Enterobacter sp. Microbacterium sp. Paenibacillus lentimorbus, Pseudomonas mevalonii, Sphingobacterium multivorum and Stenotrophomonas sp. This is the first preliminary report on the microbial diversity of SMC waste and demonstrates the presence of several species that have not been previously described in SMC, in addition to two potentially novel species within the genera Microbacterium and Stenotrophomonas. It is thereby important to examine the ecological microbe–microbe and plant– microbe interactions that are occurring between the native bacterial soil flora and those added annually (theoretically estimated at approximately 1018 cells) through the application of SMC. Such studies would be beneficial in helping to ascertain the ecological consequences involved in the disposal of SMC waste on agricultural land.

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Bacterial diversity of terra preta and pristine forest soil from the Western Amazon 

Soil Biology and Biochemistry, In Press, Uncorrected Proof, Available online 18 September 2006, Jong-Shik Kim, Gerd Sparovek, Regina M. Longo, Wanderley Jose De Melo and David Crowley

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The survey presented here describes the bacterial diversity and community structures of a pristine forest soil and an anthropogenic terra preta from the Western Amazon forest using molecular methods to identify the predominant phylogenetic groups. Bacterial community similarities and species diversity in the two soils were compared using oligonucleotide fingerprint grouping of 16S rRNA gene sequences for 1500 clones (OFRG) and by DNA sequencing. The results showed that both soils had similar bacterial community compositions over a range of phylogenetic distances, among which Acidobacteria were predominant, but that terra preta supported approximately 25% greater species richness. The survey provides the first detailed analysis of the composition and structure of bacterial communities from terra preta anthrosols using noncultured-based molecular methods.

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Possible Topics

1. You are using some pyrolysis equipment. Unfortunately the material that you are pyrolysing releases gasses which are very obnoxious. You discover that you can use a biofilter made out of straw and organisms to reduce these gasses. You want to optimise the operation and you want to find out and characterise the microorganisms involved.

a) What would you look for and why?b) What techniques would you use?c) Include a full explanation of the techniques.

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2. You are studying the operation of a sewage plant. You want to develop ways to reduce the production of greenhouse gasses from the plant. Two important green house gasses are methane and nitrous oxide. Both of these can be produced through the activities of microorganisms. What would you look for, why would you look for it and how would you look for these microorganisms? Include a full explanation of the techniques.

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3. Farmers use a lot of nitrate as fertilizer on their crops. Some people think that the constant use of nitrates eventually reduces the diversity of microorganisms in the soil which leads to soil degradation. How would you investigate this? What would you look for and why?

Include a full explanation of the techniques.