Primer Selection For any process, the primer selected for use in PCR to amplify some piece of DNA is the first point that determines what sort of information you will be working with Universal for ID, targeted for group selection, gene group targeting (NOT necessarily expressed though)

Bias Bias occurs for any reaction – some organisms will be selected over others in a way that changes their apparent abundance PCR – selects for organisms in higher abundance (not good to ID organisms at 0.x-5%, roughly) some organisms hybridize to primers faster, conditions through PCR reaction can affect…

RFLP Restriction Fragment Length Polymorphism Cutting a DNA sequence using restriction enzymes into pieces  specific enzymes cut specific places Starting DNA sequence: 5’-TAATTTCCGTTAGTTCAAGCGTTAGGACC 3’-ATTAAAGGCAATCAAGTTCGCAATAATGG Enzyme X 5’-TTC- 3”-AAG- Enzyme X 5’-TTC- 3”-AAG- 5’-TAATTT 3’-ATTAAA 5’-CCGTTAGTT 3’-GGCAATCAA 5’-CAAGCGTTAGGACC 3’-GTTCGCAATAATGG

RFLP DNA can be processed by RFLP either directly (if you can get enough DNA from an environment) or from PCR product T-RFLP (terminal-RFLP) is in most respects identical except for a marker on the end of the enzyme Works as fingerprinting technique because different organisms with different DNA sequences will have different lengths of DNA between identical units targeted by the restriction enzymes –specificity can again be manipulated with PCR primers Liu et al. (1997) Appl Environ Microbiol 63:

Electrophoresis Fragmentation products of differing length are separated – often on an agarose gel bed by electrophoresis, or using a capilarry electrophoretic separation

DGGE Denaturing gradient gel electrophoresis –The hydrogen bonds formed between complimentary base pairs, GC rich regions ‘melt’ (melting=strand separation or denaturation) at higher temperatures than regions that are AT rich. When DNA separated by electrophoresis through a gradient of increasing chemical denaturant (usually formamide and urea), the mobility of the molecule is retarded at the concentration at which the DNA strands of low melt domain dissociate. –The branched structure of the single stranded moiety of the molecule becomes entangled in the gel matrix and no further movement occurs. –Complete strand separation is prevented by the presence of a high melting domain, which is usually artificially created at one end of the molecule by incorporation of a GC clamp. This is accomplished during PCR amplification using a PCR primer with a 5' tail consisting of a sequence of 40 GC. Run DGGE animation here – from

RFLP vs. DGGE DGGE Advantages –Very sensitive to variations in DNA sequence –Can excise and sequence DNA in bands Limitations –Somewhat difficult –”One band-one species” isn’t always true –Cannot compare bands between gels –Only works well with short fragments (<500 bp), thus limiting phylogenetic characterization RFLP Advantages –Relatively easy to do –Results can be banked for future comparisons Limitations –Less sensitive phylogenetic resolution than sequencing –Each fragment length can potentially represent a diversity of microorganisms –Cannot directly sequence restriction fragments,making identification indirect

RFLP/DGGE info Grouped into sequences with SIMILARITY, not necessarily identical! Operational Taxonomic Units (OTU) is the simplest grouping Can compare these sequences to others prepared in same way (same primers for PCR, same restriction enzyme(s) ) – –

FISH Fluorescent in-situ hybridization –Design a probe consisting of an oligonucleotide sequence and a tag –Degree of specificity is variable! –Hybridize that oligonucleotide sequence to the rRNA of an organism – this is temperature and salt content sensitive –Image using epiflourescence, laser excitation confocal microscopy Technique DIRECTLY images active organisms in a sample

Fluorescent in site hybridization

10 µm DAPIFER656 B Drift Slime Streamer

Oligunucleotide design

FISH variations FISH-CARD – instead of a fluorescent probe on oligo sequence, but another molecule that can then bond to many fluorescent probes – better signal-to-noise ratio FISH-RING – design of oligo sequence to specific genes – image all organisms with DSR gene or nifH for example

FISH-MAR Combine FISH to tag specific organisms with autoradiography FISH ID’s the organism based on oligo hybridization Isotopically labelled substrates are incorporated into the organism showing metabolism

Phylogenetic Data Numerous techniques to get at the sequences: –PCR –Bacterial Artificial Chromosome (BAC) libraries –Shotgun sequencing

Clone Library

Sequencing The two generally employed methods of DNA sequence determination are the Maxam-Gilbert technique, which relies on the relative chemical liability of different nucleotide bonds, and the Sanger (dideoxy termination) method, which interrupts elongation of DNA sequences by incorporating dideoxynucleotides into the sequences.

Shotgunning Take the whole genome Break it into random fragments Sequence those fragments Re-assemble using computers to re-align portions of each fragment to get back to the entire sequence

Culturing techniques Microbiology owes it’s roots and much information we know about some organisms from culturing techniques Culturing generally attempts to develop an isolate (not always of course) Choose medium, environment (chemical and physical) – innoculate and grow… Plating or streak culturing – smear a sample so it becomes diluted…

Most Probable Number (MPN) Culturing technique that provides both cell density information and function (grouped into cells that can metabolize a certain way)

Environmental Sampling As soon as you remove organisms from their surroundings, they DO NOT just stop acting – often changing function and ecology quickly ‘Fixing’ is the technique of preserving the cells as close to their actual distribution as possible –Physical (freezing) –Chemical (additives to arrest function) –These techniques kill all, or at least most, cells Preservation – most also preserve the material you are after until you get to the lab

Materials Fixation: –Freezing – How fast? Dry ice-ethanol, liquid nitrogen –Chemical – Ethanol, Glutaraldehyde, Parafomaldehyde Preservation –Freezing –Ethanol –RNAlater –Salts, buffers

Sterile technique When should we be MOST concerned with maintaining sterile technique? –SEM samples –PCR samples –FISH samples –MPN samples –Culture samples –Shotgun samples