Molecular Methods Summary and Synthesis

Review

How can techniques developed by molecular biologists be used to answer ecological

questions?

Nucleic acids (DNA and RNA) are present in all calls – Bacteria, Archaea and Eukaryotes. Molecular techniques use nucleic acids to identify species and determine relationships without having to grow or culture the microorganisms.

Ribosomal RNA (rRNA) and the genes that code for it (rDNA) have both highly conserved and variable regions, which makes this molecule useful for this type of comparative analysis.

One major limitation of this method is that they can identify the DNA of the microbes present, but not whether those microbes were living and active at the time of collection.

DNA extraction1. Lyse cell membrane

a. Chemically detergentb. Physically bead beating

2. Pellet cell membrane, proteins and other cell parts while DNA stays in solution

3. Remove other inhibitors from DNA

4. Mix DNA with acid and salt stick to filter

5. Wash filter-bound DNA several times with alcohol

6. Elute DNA off membrane with pH 8, low-salt buffer

Your DNA

L RB MC GG AS BP LS

Genomic DNA = the sum total of all DNA from an organism or a community of organisms

Ribosomal RNA

Structural molecule involved in protein synthesisHas a large subunit and a small subunit

Small subunit has variable regions and conserved regionsUsed for phylogenetic comparisons (Who’s there?)

Why are SSU rRNA genes so widely used in biodiversity studies?

• Ubiquitous occurrence among all living thingsUbiquitous occurrence among all living things• Functional uniformityFunctional uniformity• Absence of lateral gene transferAbsence of lateral gene transfer• Possession of conserved and variable regions Possession of conserved and variable regions

which allow for nucleotide base pair alignments which allow for nucleotide base pair alignments between closely and distantly related organismsbetween closely and distantly related organisms

• Large data baseLarge data base

PolymeraseChain Reaction

What can molecular biology tell us about ecology?

• Diversity of organisms (who’s there?) – how many groups of organisms are inhabiting a system? Which groups are co-occurring? How related are they to organisms living elsewhere?

• (for instance, bacteria related to “hyperthermophiles” have been found in the Antarctic…)

• Activity of organisms (what are they doing?) – which genes do the organisms possess? Metal degradation, methanogenesis, arsenic utilization? Which genes are the organisms actively using at the moment of collection?

• often organisms carry genes in their genome that they never use, which can be tricky for molecular biologists

Aerobic Bacteria and Eukaryotes - primarily using Oxygen for energy-Oxidize sulfur that drifts up from below- some are flagellated, motile, or form mats

Anaerobic bacteria and Archaea-Fermentation- Anaerobic respiration (Sulfur reduction)

Bacteria and Eukaryotes - use Oxygen and/or oxygenated energy sources like Nitrate (NO3)

“A”

500400

AS G M B R L NEG

Nanoarchaea?

Bacteria

1.5KB

Archaea

1 KB

G A M L R B

Dissimilatory bisulfite reductase

BP RB MC GG LS AS

mcrA

Methyl coenzyme M reductase (subunit A)

Catalyzes the reduction of a methyl group bound to coenzyme-M, with the concomitant release of methane. This enzyme complex is thought to be unique to, and ubiquitous in, methanogens.

LLS RB BP MC GG AS NEG

2 KB

EUKARYOTES

DGGE

• DNA is negatively charged• will migrate through gel towards positively charged anode

• If gel contains ‘denaturant’, H-bonds between strands will start to break apart• based on sequence (A-T bonds will go first….)

• As strands denature their migration slows down

• Each unique sequence denatures differently – each stops migrating at a different place

Ladder

10μl

A

15μl 20μl 10μl 15μl 20μl Ladder

B

Cloning

T-RFLP: Terminal restriction fragment length polymorphism

RFLP: Restriction fragment length polymorphism

Organism AOrganism AOrganism BOrganism BOrganism COrganism COrganism DOrganism D

Organism AOrganism A

Organism BOrganism BOrganism COrganism C

Organism DOrganism D

Distance Matrix

Maximum Parsimony

Maximum Likelihood

C AT GT A GC TG C C TC C C A A

C -T GT - GC CG T C TT T C G A

G AT G- A GC TG C C TC C C A A

G -T GT A CC TG C C TC C C A A

Euglenoids

FUNGI

Sulfolobus Thermoplasma

Plant Mitochondria

CyanobacteriaPlant Chloroplasts

Myco- plasma

Red Algae

Enterobacteria

MethanobacteriaHalobacteria

BACTERIA

PLANTAE

ANIMALIA

STRAMENOPILES

ALVEOLATES

Entamoebae

Kinetoplastids

Heterolobosea

Slime Molds

Physarum

DiplomonadsTrichomonads

Microsporidians

EUKARYA

ARCHAEA

Agrobacterium

Nitrogenous base

Sugar

Phosphate group

Phosphodiester bond

•Right handed double helix

•Stabilized by H-bonds between base pairs

•Hydrophobic bases inside, hydrophilic phosphate groups outside

Topoisomerase: introduces negative supercoil in DNA strand

Helicase: unwinds DNA helix by breaking hydrogen bonds between base pairs – usually at the weaker A-T bonds.

RNA Primase: produces short pieces of RNA – like primers – that are recognized by DNA polymerase to start replication.

DNA polymerase: recruits nucleotides and copies DNA strand in complementary fashion starting with RNA primers.

Exonuclease: removes RNA primers. DNA polymerase fills in gaps.

DNA REPLICATION

New strand formed 5’ > 3’

DNA replication

1. DNA replication begins at the origin of replication.2. DNA helicase unwinds double-stranded DNA.3. Topoisomerases stabilize single-stranded DNA.4. Primase synthesizes and attaches RNA primers to the single DNA strand.5. DNA polymerase adds new nucleotides to a growing DNA

strand.6. Short Okazaki fragments form.7. DNA ligase links together Okazaki fragments.