DNA sequencing using fluorescence background electroblotting membrane

Patent · Wed Jan 01 00:00:00 EST 1992

OSTI ID:868284

Caldwell, Karin D ^[1]; Chu, Tun-Jen ^[1]; Pitt, William G ^[2]

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1. Salt Lake City, UT
2. Orem, UT

A method for the multiplex sequencing on DNA is disclosed which comprises the electroblotting or specific base terminated DNA fragments, which have been resolved by gel electrophoresis, onto the surface of a neutral non-aromatic polymeric microporous membrane exhibiting low background fluorescence which has been surface modified to contain amino groups. Polypropylene membranes are preferably and the introduction of amino groups is accomplished by subjecting the membrane to radio or microwave frequency plasma discharge in the presence of an aminating agent, preferably ammonia. The membrane, containing physically adsorbed DNA fragments on its surface after the electroblotting, is then treated with crosslinking means such as UV radiation or a glutaraldehyde spray to chemically bind the DNA fragments to the membrane through said smino groups contained on the surface thereof. The DNA fragments chemically bound to the membrane are subjected to hybridization probing with a tagged probe specific to the sequence of the DNA fragments. The tagging may be by either fluorophores or radioisotopes. The tagged probes hybridized to said target DNA fragments are detected and read by laser induced fluorescence detection or autoradiograms. The use of aminated low fluorescent background membranes allows the use of fluorescent detection and reading even when the available amount of DNA to be sequenced is small. The DNA bound to the membrances may be reprobed numerous times.

• Research Organization: Univ. of Utah, Salt Lake City, UT (United States)
• DOE Contract Number: FG02-88ER60700
• Assignee: University of Utah (Salt Lake City, UT); Brigham Young University (Provo, UT)
• Patent Number(s): US 5112736
• OSTI ID: 868284
• Country of Publication: United States
• Language: English

Related Subjects

dna
sequencing
fluorescence
background
electroblotting
membrane
method
multiplex
disclosed
comprises
specific
base
terminated
fragments
resolved
electrophoresis
surface
neutral
non-aromatic
polymeric
microporous
exhibiting
modified
contain
amino
polypropylene
membranes
preferably
introduction
accomplished
subjecting
radio
microwave
frequency
plasma
discharge
presence
aminating
agent
ammonia
containing
physically
adsorbed
treated
crosslinking
means
radiation
glutaraldehyde
spray
chemically
bind
smino
contained
bound
subjected
hybridization
probing
tagged
probe
sequence
tagging
fluorophores
radioisotopes
probes
hybridized
target
detected
laser
induced
detection
autoradiograms
aminated
fluorescent
allows
reading
available
amount
sequenced
membrances
reprobed
numerous
times
fluorescence detection
microwave frequency
plasma discharge
porous membrane
dna fragments
dna sequencing
laser induced
target dna
dna fragment
multiplex sequencing
surface modified
chemically bound
microporous membrane
induced fluorescence
specific base
chemically bind
wave frequency
fluorescent detection
frequency plasma
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