Download DNA polymerase

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 extraction
1. Lyse cell membrane
a. Chemically  detergent
b. 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 synthesis
Has a large subunit and a small subunit
Small subunit has variable regions and conserved regions
Used for phylogenetic comparisons (Who’s there?)
Why are SSU rRNA genes so widely
used in biodiversity studies?
•
•
•
•
Ubiquitous occurrence among all living things
Functional uniformity
Absence of lateral gene transfer
Possession of conserved and variable regions
which allow for nucleotide base pair alignments
between closely and distantly related organisms
• Large data base
Polymerase
Chain
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 cooccurring? 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
Bacteria and Eukaryotes
- use Oxygen and/or oxygenated energy
sources like Nitrate (NO3)
Anaerobic bacteria and Archaea
-Fermentation
- Anaerobic respiration
(Sulfur reduction)
“A”
Nanoarchaea?
AS
500
400
G
M
B
R
L
NEG
Bacteria
1.5KB
Archaea
G
1 KB
A
M
L
R
B
Dissimilatory bisulfite reductase
BP
RB
MC
GG
LS
AS
Methyl coenzyme M reductase (subunit A)
mcrA
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.
EUKARYOTES
LLS
2 KB
RB
BP
MC
GG
AS
NEG
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
A
Ladd
er
10μl
15μl 20μl
B
10μl 15μl 20μl
Ladd
er
Cloning
T-RFLP: Terminal restriction fragment length polymorphism
RFLP: Restriction fragment length polymorphism
Organism A
Organism B
Organism C
Organism D
C T
T A C G G C C T C C T A C A G
C T
T
- C G G T C C T T T
- C G G
A
G T
-
A C G G C C T C C T A C A G
A
G T
T A C C G C C T C C T
Organism A
- C A G
A
A
Organism D
Organism B
Organism C
Distance Matrix
Maximum Likelihood
Maximum Parsimony
STRAMENOPILES
EUKARYA
PLANTAE
ALVEOLATES
ANIMALIA
Red Algae
BACTERIA
Slime Molds
MycoEntamoebae
Plant Chloroplasts plasma FUNGI
Heterolobosea
Cyanobacteria
Physarum
Agrobacterium
Kinetoplastids
Euglenoids
Plant Mitochondria
Microsporidians
Enterobacteria
Trichomonads
Diplomonads
Sulfolobus
Thermoplasma
Halobacteria
Methanobacteria
ARCHAEA
Nitrogenous base
Phosphodiester bond
Phosphate group
Sugar
•Right handed double helix
•Stabilized by H-bonds between base
pairs
•Hydrophobic bases inside,
hydrophilic phosphate groups
outside
DNA REPLICATION
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.
New strand formed 5’ > 3’
Exonuclease: removes RNA
primers. DNA polymerase fills in
gaps.
DNA replication
1.
2.
3.
4.
DNA replication begins at the origin of replication.
DNA helicase unwinds double-stranded DNA.
Topoisomerases stabilize single-stranded DNA.
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.