Molecular Biology

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Molecular Biology

• Materials Methods 4
• Sequencing Synthesis

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Venter et al. 2001 Science gt 100 authors!
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Science 2006
X-Prize 10 million
Science 2005
Science 2006
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Pyrosequencing detects pyrophosphate
(P-P) released from polymerization
One of several novel sequencing methods in
experimental stage
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Pyrosequencing set-up steps
Whole DNA
sonicate
short (300-500 bp) dsDNA
denature attach to bead
short ssDNA
copy by PCR on bead
many copies of same DNA on bead
distribute 1 bead into each well
add dNTPs one at a time
new DNA strand grows when correct dNTP is added
pyrosequencing reactions in wells produces light
detected by CCD camera
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Pyrosequencing plate measures 70 x 75 mm, has 1.6
million wells Each DNA strand can be sequenced
for ca. 100 bases
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