DNA-RNA unit
ATP
RNA
Nucleoid
• Single strand of DNA, usually circular, usually
looks like a big ball of messed up twine…
• Size – smallest organism yet discovered
(Nanoarchaeum equitans) 490,889 base pairs; e.
coli 4.7 Mbp, most prokaryotes 1-6 million base
pairs (1-6 MBp); Humans 3300 MBp
•DNA is around 1000 mm long in
bacteria, while the organism is on the
order of 1 mm long – special
enzymes called gyrases help coil it
into a compact form
Ribosomes
• Ribosomal RNA is single stranded
• RNA is a single stranded nucleic acid
– mRNA- messanger RNA – copies information from
DNA and carries it to the ribosomes
– tRNA – transfer RNA – transfers specific amino acids to
the ribosomes
– rRNA – ribosomal RNA – with proteins, assembles
ribosomal subunits
DNA is transcribed to produce mRNA
mRNA then translated into proteins.
RNA and protein construction
• The nucleotide base sequence of mRNA is encoded
from DNA and transmits sequences of bases used to
determine the amino acid sequence of the protein.
• mRNA (“Messenger RNA”) associates with the ribosome
(mRNA and protein portion).
• RNA (“Transfer RNA”) also required
• Codons are 3 base mRNA segments that specify a
certain amino acid.
• Most amino acids are coded for by more than one
codon: degenerage genetic code.
• Translation ends when ribosome reached “stop codon”
on mRNA.
Transcription
RNA polymeraze takes the DNA and temporarily unwinds it, templates the
transfer RNA from that, using ribonucleoside triphosphates to assemble…
Translation
• mRNA is coded for one or more specific
amino acids and moves to the ribosome to
assemble amino acids into proteins
• On mRNA, codons are 3 bases, coded to
specific amino acids
• On tRNA, the anticodon
latches to the codon
on the mRNA
Protein Formation
•
The ‘code’ on
mRNA
determines the
sequence of
protein assembly
rRNA
• Ribosomes are made of proteins and rRNA, the
tRNA and mRNA come to it and assemble the
proteins
• rRNA plays a structural role, serving as a
support for protein construction, and a functional
role
• rRNA consists of two subunits, one 30S in size
(16S rRNA and 21 different proteins), one 50S in
size (5S and 23S rRNA and 34 different
proteins). The smaller subunit has a binding site
for the mRNA. The larger subunit has two
binding sites for tRNA.
Microbial Genomics
• Study of the gene sequence of an
organism
• Tree of Like – Ernst Haekel, 1866
Tree of Life
•
Global phylogeny of fully sequenced organisms. The phylogenetic tree has its basis in a cleaned and concatenated alignment of 31
universal protein families and covers 191 species whose genomes have been fully sequenced (14). Green section, Archaea; red,
Eukaryota; blue, Bacteria. Labels and color shadings indicate various frequently used subdivisions. The branch separating Eukaryota and
Archaea from Bacteria in this unrooted tree has been shortened for display purposes.
http://www.sciencemag.org/cgi/content/full/311/5765/1283/FIG2
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???
–
–
–
–
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
•
Looking to compare similarity (in base 4) of sequences
1.
2.
3.
4.
•
ATTGGCCACG
ATTCGCCTCG
TGGCGCCTTT
ATTGGGCACG
Determine the ‘degree of similitude’ between these and
represent that graphically
1
3
4
2
•
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
OK – How do we get genetic data?
• DNA extraction
• Since DNA is more stable, easier to extract and
preserve than is rRNA
• Need to get the DNA ‘out’ of the organism
– Bead beating
– Chemical
– Thermal
• Purify this material through chemical and physical
extraction/filtration
• A multitude of techniques exist to do this – often
specific for the type of sample
• Why??
Now that you have the DNA…
• For most techniques, even for the high cell
densities of biofilms or cultures, there is nowhere
near enough material to actually analyze
• While the genetic code was known about for many
years, it was a special discovery by a surferscientist, Kary Mullis, that kick-started modern
molecular biology – PCR (for which he received
the 1993 Nobel Prize in Chemistry)
• Based on the thermal tolerance of a DNA
polymerase first isolated from the thermophile
Thermus aquaticas (thus Taq polymerase)
• The first part of Mullis's idea was to use pairs of short, single-stranded DNA
pieces as probes to bracket the exact DNA sequence in which you happened
to be interested. You could then cleverly employ iterative biochemical
processes to get that DNA sequence to 'reproduce the hell out of itself'. You
could start with the tiniest samples of even impure DNA and wind up within
hours with a test-tube full of whatever gene or DNA sequence you had
targeted. 'I would be famous,' Mullis recalls thinking at the moment of
discovery, 'I would get the Nobel Prize.'
• The discovery of PCR is here presented as a great American epiphany - its
recipient struck by a flash of inspiration on the road, not to Damascus, but to
Mendocino. Sometimes Mullis says the creative spirit came on an evening in
April of 1983, sometimes in May. Anyway, the buckeyes were in bloom;
Mullis's little silver Honda Civic was purring through the vineyards and
redwoods of the Anderson Valley; and his mind wandered. Life is sweet, he
thought: 'I am a big kid with a new car and a full tank of gas. I have shoes
that fit. I have a woman sleeping next to me and an exciting problem, a big
one.' At mile-marker 46.58 on Highway 128 - he had both the presence of
mind and the sense of history to note the exact spot, if not the month - the
epiphany arrives. 'Holy shit,' Mullis cries out, and his girlfriend almost, but
not quite, wakes up. He pulls the Honda to the side of the road to write down
his ideas and check his calculations. Within feverish minutes, the problem is
solved, and Mullis is left with the mop-up operation of getting PCR actually
to work. This takes almost two years, and the original report was famously
rejected by both Nature and Science. Mullis was not fazed: '"Fuck them," I
said.'
Excerpts from ‘Dancing Naked in the Mind Field’ by Kary Mullis
PCR - Polymerase Chain Reaction
• PCR is an in vitro technique for the amplification of a region of DNA
which lies between two regions of known sequence.
• PCR amplification is achieved by using oligonucleotide primers.
– These are typically short, single stranded oligonucleotides which are
complementary to the outer regions of known sequence.
• The oligonucleotides serve as primers for DNA polymerase and the
denatured strands of the large DNA fragment serves as the template.
– This results in the synthesis of new DNA strands which are
complementary to the parent template strands.
– These new strands have defined 5' ends (the 5' ends of the
oligonucleotide primers), whereas the 3' ends are potentially
ambiguous in length.
Primer selection
• Primer is an oligonucleotide sequence – will target
a specific sequence of opposite base pairing (A-T,
G-C only) of single-stranded nucleic acids
• For example, there is a
– ¼ chance (4-1) of finding an A, G, C or T in any given DNA
sequence; there is a
– 1/16 chance (4-2) of finding any dinucleotide sequence (eg.
AG); a
– 1/256 chance of finding a given 4-base sequence.
• Thus, a sixteen base sequence will statistically be
present only once in every 416 bases (=4 294 967
296, or 4 billion): this is about the size of the human or
maize genome, and 1000x greater than the genome
size of E. coli.
Primer Specificity
• Universal – amplifies ALL bacterial DNA
for instance
• Group Specific – amplify all denitrifiers for
instance
• Specific – amplify just a given sequence
Forward and reverse primers
• If you know the sequence targeted for
amplification, you know the size which the
primers should be anealing across
• If you don’t know the sequence… What
do you get?
DNA Polymerase
• DNA Polymerase is the enzyme responsible for
copying the sequence starting at the primer from
the single DNA strand
• Commonly use Taq, an enzyme from the
hyperthermophilic organisms Thermus aquaticus,
isolated first at a thermal spring in Yellowstone
National Park
• This enzyme is heat-tolerant  useful both because
it is thermally tolerant (survives the melting T of
DNA denaturation) which also means the process is
more specific, higher temps result in less mismatch
– more specific replication
•
http://ocw.mit.edu/NR/rdonlyres/Civil-and-Environmental-Engineering/1-89Fall-2004/321BF8FF-75BE-4377-8D74-8EEE753A328C/0/11_02_04.pdf