Advanced Environmental Biotechnology II Week 05 – DNA fingerprinting of microbial communities

Based on the chapter 3 DNA fingerprinting of microbial communities Andreas Felske and A.Mark Osborn   [from: Molecular Microbial Ecology BIOS Advanced Methods. (2005) Osborn, A. Mark.; Smith, Cindy J. Eds. Taylor & Francis Routledge]

3.1  Introduction   Chance to study microbial communities and diversity by analysis of nucleic acids directly extracted from environmental samples

Variety of DNA fingerprinting techniques has allowed investigation of - variation in microbial communities, in particular by study of ribosomal RNA and - variation between individual functional genes.

MAR=microautoradiography, terminal restriction fragment length polymorphism (T-RLFP), denaturing/ thermal gradient gel electrophoresis (D/TGGE) and single strand conformation polymorphism (SSCP)

DNA fingerprinting → rapid generation of characteristic patterns for entire microbial communities from large numbers of environmental samples.

→ very simplified representation of the microbial community, DNA fingerprinting → useful for monitoring changes in microbial communities in time and space with minimal effort. → very simplified representation of the microbial community, defined by the specificity of the oligonucleotide primers and by the preferential amplification of DNA fragments during PCR.

Separating complex PCR products into fingerprints → single bands for predominant sequences → rare sequences may often remain undetected.

In very complex environmental communities few predominant species → a minority thousands of rare species → most of the biomass but no signal in the fingerprint.

Loss of information a drawback of any PCR-based investigation of environmental samples.

Fingerprinting avoids some problems of PCR

First DNA fingerprinting approach to be successfully applied to microbial ecology was denaturing gradient gel electrophoresis (DGGE) The vast majority of studies applying DNA fingerprinting to microbial communities are focused on ribosomal RNA.

DGGE led to other profiling approaches eg: temperature gradient gel electrophoresis (TGGE) single strand conformation polymorphism (SSCP) analysis terminal restriction fragment length polymorphism (T-RFLP) and length heterogeneity PCR (LH-PCR)

DGGE and TGGE are based on the differential melting of GC-rich DNA stretches in the amplified DNA molecules.

SSCP separates on the basis of different melting behavior of the secondary structures of single-stranded DNA.

T-RFLP generates DNA fragment length variations via the presence of restriction sites.

LH-PCR uses the different length of DNA stretches in hyper-variable regions of the target gene and in particular for ribosomal RNA.

3.1.1 DGGE Single strand (ss) DNA migrates more slowly than double strand (ds) DNA during electrophoresis.

Denaturing gradient gel electrophoresis Many studies use denaturing gradient gel electrophoresis (DGGE). It can separate DNA-fragments of the same size but with different nucleic acid sequences.

From: Oil Pollution and Its Environmental Impact in the Arabian Gulf Region  M. Al-Azab, W. El-Shorbagy, S. Al-Ghais. Elsevier, 2005

Band patterns show the genetic biodiversity of the sample. The number of bands equals the number of dominant species. This can give a good idea of what is in a microbial community. It is best with sequencing and phylogenetic analysis of the bands.

Diagram showing steps of DGGE

(A) DNA is extracted from the original sample, in this case as granular sludge from a UASB reactor.

(B) The 16S rRNA gene is partly amplified by PCR with primers to give a mixture of DNA fragments, all of the same length.

(C) The DNA mixture is separated by denaturant gradient electrophoresis on a gel with an increasing urea/formamide gradient.

The DNA moves towards the positive pole and stops when it reaches its denaturant force (Tm). Every band on the gel means a different microorganism.

(D) The bands can be cut from the gel and the DNA extracted and sequenced.

(E) Comparison of the sequences with a 16S rDNA database shows the phylogeny of the microorganism.

DGGE is good to find the main members of a microbial community with some idea of what they are related to. This method has been used for soil, bacterioplankton, hot springs, oceans, etc.

DGGE is used with other methods, for example with in situ hybridization in the study of sulfate reducing bacteria or phosphorous elimination.

The most important application of DGGE is studying dynamic changes in microbial communities, especially when many samples have to be processed.

DGGE has been used to: study differences between mesophilic and thermophilic reactors.

DGGE has been used to: analyze the changes observed in the bacterial diversity of an anaerobic digester for treating urban solid waste.

DGGE has been used to: study the changes in bacterial communities in a continuous stirred tank reactor (CSTR) in response to dilution rate.

DGGE (or TGGE: temperature gradient gel electrophoresis) is the most used genetic fingerprinting technique in molecular ecology.

DGGE Advantages It gives rapid and simple monitoring of how microbial populations change in space and time if just band patterns are considered.

DGGE Advantages It is relatively easy to get an overview of the main species of an ecosystem.

DGGE Advantages It is good for analysis of a large number of samples. It can be used for many more samples than cloning.

DGGE Disadvantages Extraction and amplification of representative genomic DNA can be difficult (as in cloning).

DGGE Disadvantages The DNA copy number can be very different (as in cloning). The intensity the bands obtained on a DGGE gel may vary.

DGGE Disadvantages The number of detected bands is usually small, which suggests: the number of identified species is also small; the bands correspond to the major species in the original sample.

DGGE Disadvantages The sequences of the bands obtained from a gel correspond to short DNA fragments (200–600 bp), and so phylogenetic relations are less reliable than with cloning of the whole 16S rRNA gene. Short sequences are less useful for designing new specific primers and probes.

3.1.2 TGGE TGGE is like DGGE but the increasing denaturing force is an increase in gel temperature towards the anode. A high concentration of chemical denaturing agents is included in the gel mix.

The theory behind the separation of DNA molecules via TGGE is exactly the same as for DGGE.

3.1.3 SSCP analysis The electrophoretic mobility of a single-stranded DNA molecule in a non-denaturing gel is dependent on its structure and size.

Single-stranded DNA molecules → secondary structure by base pairing between nucleotides within a single strand. Depend on length of the DNA stretch and location and number of regions of base pairing.

Especially prominent in rDNA fragments → SSCP analysis is very well suited to the rDNA-based analysis of microbial communities. A single nucleotide change may alter the conformation of a ssDNA molecule and will allow two DNA fragments that differ in only one nucleotide to be distinguished when electrophoresed in non-denaturing polyacrylamide gels due to mobility difference between the molecules.

No GC clamp primers, gradients, or specific apparatus is needed. A significant limitation of SSCP is the formation of more than one band from a single sequence.

Often three bands can be detected, one from each of the denatured single-stranded DNA molecules and a third band from undenatured dsDNA molecules. Even more bands may appear if different conformations of one product are possible.

A second disadvantage of SSCP is the high rate of DNA re-annealing during electrophoresis ↓ signal intensity for the ssDNA bands.

↑ concentration of PCR product that is loaded on a SSCP gel ↑ the effects of renaturation will spoil the display of resolved products.

Schwieger and Tebbe amplified 400 bp fragments of the bacterial 16S rDNA using universal primers, for which one primer was phosphorylated at the 5′-end. Phosphorylated strand of the PCR products can then be removed by λ-exonuclease treatment.

→ resulting signal will be produced from only one strand and any possible re-annealing or heteroduplex formation will be avoided.

3.1.4 T-RFLP analysis Also known as amplified rDNA restriction analysis (ARDRA) Easy comparison of rDNAs from bacterial isolates or clone libraries.

PCR products are obtained by using universal 16S rDNA primers, and the product is digested with restriction enzymes with 4 bp recognition sites.

The 16S rDNA PCR products from different bacteria, provided the same primers were used, would show only limited variation in length.

However, the restriction sites may be found at very different positions within 16S rDNA sequences from different bacteria and so they cut the PCR product into two or more fragments of different length.

Locate restriction sites within the 16S rDNA, which is sequence specific and therefore potentially taxon specific.

Sequence databases may be searched for taxon-specific restriction sites and experiments can be readily customized by selecting the appropriate primers and restriction enzymes.

Since PCR products amplified from different bacterial 16S rDNA will be split into two or more fragments, the RFLP fingerprint would be even more complex than that of the original PCR product. eg taxa represented by six restriction fragments could be overestimated compared to those that yield only two fragments.

Add a fluorescent dye to one of the primers that can be detected by the fluorescence-based DNA sequencer/genetic analyzer being used. The primer will be built into the PCR product, but following restriction digestion only the terminal fragment containing the labeled primer will be detected by an automated DNA sequencing instrument. → terminal RFLP (T-RFLP)

T-RFLP can be used with DNA from complex microbial communities and provides a valuable method to produce fingerprints of the general microbial community composition.

3.1.5 LH-PCR (length heterogeneity PCR) Length heterogeneity analysis of PCR amplified genes (LH-PCR) uses naturally occurring differences in the lengths of amplified gene fragments.

Generally, strong heterogeneity seems to be most common in the apical helices of ribosomal molecules, i.e. those ending in a hairpin loop.

The sizes of the fragments on the polyacrylamide gel after electrophoresis can be compared against 16S rRNA gene databases to specify microbial groups that may correspond in size to the size of the fragments.

PCR amplification of a small part of the target gene with a labeled primer and then electrophoresis of the labeled product on an automated fluorescence-detection-based sequencing device.

An internal standard labeled with a different fluorescent dye is run together with the sample to allow determination of fragment length.

Universal primers between E Universal primers between E. coli positions 8 and 355 includes the highly variable regions V1 and V2 and yielded fragments of 312–355 bp in length, and identified up to 23 distinct length heterogeneity variants.