Download PCR - Polymerase Chain Reaction

Microbial Genetics
• As we have looked at, information for all
cellular function is from DNA, mRNA
carries that info to ribosomes, rRNA codes
for proteins constructed at ribosomes
• So – in an environmental/geological
context, what is that info used for??
Genomic info Gives us:
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Changing the genetic code:
• Mutation: Change in at least one nucleotide
– Point mutations: 1 nucleotide change
– Framesift mutations: base-pair change
• Think on how sequence is ‘read’
– ATTGGCCATAGG
– ATTGGCCATAGG  Codon grouping with a start and
stop coding
– Frameshift  changes that mess up codon groups, mess
up start/stop info – completely change the protein…
– Result  Silent (codons are degenerate – amino acids
coded by several possible sequences), Missence (amino
acid substitution), Nonsense (shorter or longer protein)
Mutation agents:
• ‘Natural’ error in replication
• Chemical agent:
– acids – esp. nitrous acid by modifying structure of adenine
– Base analog – organic base similar enough to substitute in
– Mutagens – cause base-pair problems or disruption of
chain
• Physical agent:
– UV light – disrupts base pairing – thymine dimerization (TT bonds form)
– Radiation damage – high energy particles disrupt
electronic structure and bonding
• Viruses and gene transfer – adding snippets of
‘foreign’ code
Genetic repair
• Organisms have the machinery to repair
damaged DNA
• Some organisms have adapted to handle
doing this often, as is the case for a
particular bacteria that can survive in
abnormally high radiation: Deinoccocus
radiodurans (can survive 1.5 million rads –
3000x what would kill us)
Microbial Identification
• All organisms have unique genetic codes
describing their composition and function
• The diffference between these codes is a
measure of how similar these things are to
each other
• IF all organisms evolved from a common
ancestor, then all codes of current life
came FROM that common ancestor
• – this idea is the study of phylogentics
Construction of phylogenies
• What piece of the genetic code is most
appropriate for determining these relationships –
what should that part of DNA be responsible
for???
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Homologous function (common to all organisms)
Larger is better (more info)
Rate of mutation accumulation relevant to evolution
Does it all make sense – i.e., when you look at the
phylogeny does it make sense? – function and
evolution??
Comparative sequence analysis
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Looking to compare similarity (in base 4) of sequences
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ATTGGCCACG
ATTCGCCTCG
TGGCGCCTTT
ATTGGGCACG
Determine the ‘degree of similitude’ between these and
represent that graphically
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Literally hundreds of ways to do this…
What piece of DNA do we use
• 16S rRNA coding – info that is responsible
for the 16S subunit in ribosomes
– All bacteria and archea have 70S ribosomes
with a 16S subunit
– All bacteria and archaea need this to make
proteins
– Some parts accumulate mutation slow, other
parts fast (evolution resolved over long time
but also with good resolution)
After original work of Carl Woese and colleagues
Clone Library
•
http://ocw.mit.edu/NR/rdonlyres/Civil-and-Environmental-Engineering/1-89Fall-2004/321BF8FF-75BE-4377-8D74-8EEE753A328C/0/11_02_04.pdf
http://www.ifa.hawaii.edu/UHNAI/NAIweb/presentations/astrobiol6.pdf
DNA Sequencing
DNA sequencing reactions are just
like the PCR reactions for replicating
DNA. The reaction mix includes the
template DNA, free nucleotides, an
enzyme (usually a variant of Taq
polymerase) and a 'primer' - a small
piece of single-stranded DNA about
20-30 nt long that can hybridize to
one strand of the template DNA. The
reaction is initiated by heating until
the two strands of DNA separate,
then the primer sticks to its intended
location and DNA polymerase starts
elongating the primer. If allowed to go
to completion, a new strand of DNA
would be the result. If we start with a
billion identical pieces of template
DNA, we'll get a billion new copies of
one of its strands.
http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html
• Dideoxynucleotides: We run the
reactions, however, in the
presence of a
dideoxyribonucleotide. This is just
like regular DNA, except it has no
3' hydroxyl group - once it's added
to the end of a DNA strand, there's
no way to continue elongating it.
Now the key to this is that MOST
of the nucleotides are regular
ones, and just a fraction of them
are dideoxy nucleotides....
http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html
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End up with billions of strands, with
representatives of every length…
http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html
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 and there's no
problem. MOST of the time after
adding a T, the enzyme will go
ahead and add more nucleotides.
However, 5% of the time, the
enzyme will get a dideoxy-T, and
that strand can never again be
elongated. It eventually breaks
away from the enzyme, a dead end
product.
Sooner or later ALL of the copies
will get terminated by a T, but each
time the enzyme makes a new
strand, the place it gets stopped will
be random. In millions of starts,
there will be strands stopping at
every possible T along the way.
• Gel electrophoresis can be used
to separate the fragments by
size and measure them.
http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html
• Tag the terminal
bases with a
fluorescent tag
specific to each base
• Now can separate
based on size AND
see which base that
size is terminated
with…
• Start (GCGA) is
based on a primer!
http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html
• Gel electrophoresis separates the
products with respect to size, a
fluorescence detector ID’s the terminal
base
• Yields the sequence…
• Works great starting from a specific place
(16S rDNA for example…)
http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html
How to get more of the gene?
• Several different methods developed to
get more of the genome (ideally the
complete genome)
• Pyrosequencing (454)
• Randominzed BAC libraries
• Shotgun sequencing
• Get many fragments of the genome, trick
is to find overlap and reassemble!!
http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html