DNA mobility modifier

Details DNA mobility modifier DNA mobility modifier

2002-07-16

2004-04-20

Riley, Jezia (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S091200, C536S023100, C536S024300, C536S025300

Reexamination Certificate

active

06723515

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a composition for modifying the mobility of DNA in a sieving or non-sieving matrix, and to methods of using the composition for determining the sequence of a target polynucleotide.
BACKGROUND OF THE INVENTION
Miniaturized electrophoresis systems have the potential to substantially increase the speed and throughput of automated DNA sequencing while reducing the overall cost per base. Current miniaturized electrophoresis systems, in particular capillary electrophoresis, continue to rely upon the use of viscous polymer solutions as “gels” that provide a physical separation of DNA fragments according to chain length.
Unfortunately, gel-based methods for DNA sequencing have a maximum read lengths of about 1000 bases, with a typical read length being at most about 550 to about 650 bases. Since engineered DNA polymerases can produce more than 2500 bases of sequence per Sanger sequencing reaction, even optimized sequencing gels essentially “hide” or “lose” about 1500 bases of information per reaction. Moreover, the replacement of viscous polymer gels from chip microchannels is difficult, if not impossible, thus preventing the use of automated chip-based gel electrophoresis systems for multiple, consecutive DNA sequencing analysis.
In addition to the above-described read-length limitations of gel electrophoresis, there is another drawback to the use of gels that makes them particularly inconvenient for capillary and microchip geometries. A reusable sequencing chip or capillary array must allow gels to be replaced before each run to eliminate sample carry-over and avoid irreproducibilities and current failures that may result from chemical- and/or field-induced gel breakdown. Unfortunately, no replaceable, high-performance sequencing gel appropriate for the microchannels on CE chips is currently available. And since sequencing gels cannot be replaced from chip microchannels, each chip is essentially a single-use device, an outcome that will greatly increase associated operating costs and decrease the convenience of miniaturized DNA sequencers.
Moreover, the sequencing gels used in experimental microchips currently are typically polymerized in situ, which typically yields low-performance gels because acrylamides generally do not polymerize to high molecular weights or yield greater than 85% conversion, i.e., polymerization, in a confined microchannel. While extremely “large” channels may be fabricated onto the chip (e.g., 100 &mgr;m wide×90 &mgr;m deep), which allows viscous gels to be forced into the channels under pressure, these large channels, however, result in broadened DNA bands and/or reduced separation efficiency.
Therefore, there is a need for a method for electrophoretic DNA sequencing that does not demand the use of high-viscosity physical gels or cross-linked chemical gels. There is also a need for novel sequencing technologies that will substantially increase the speed and throughput of automated sequencing instruments while reducing the overall cost per base.
SUMMARY OF THE INVENTION
The present invention provides a compound and methods for using the same in electrophoretic separation of binding polymers. In particular, the present invention provides a polyamide compound comprising at least one hydrophilic C
1
-C
10
hydrocarbyl substituent on an amide nitrogen atom and methods for using the polyamide in electrophoretic separation of binding polymers in a non-sieving liquid medium.
One aspect of present invention provides a polyamide of the formula:
where
L
1
is selected from the group consisting of H, amide protecting groups and moieties of the formula:
each X is independently an amino acid side-chain residue or of the formula —CH
2
SL
2
or —(CH
2
)
3
NL
3
R
4
;
each L
2
is independently a thiol protecting group or a moiety of the formula:
each L
3
is independently H, an amine protecting group, an &agr;, &bgr;-unsaturated carbonyl moiety or a conjugate moiety of the formula:
provided at most one and only one L
3
is the conjugate moiety;
each L
4
is independently H, amide protecting groups or the moiety of the formula:
each R is independently —CH
2
— or
each R
1
is independently H, a protecting group or C
1
-C
10
hydrocarbyl;
each of R
2
and R
3
are independently H, C
1
-C
6
alkyl, or an amide protecting group;
each R
4
is independently H, an amine protecting group, or C
1
-C
6
alkyl, provided at least one of L
3
or R
4
on the same nitrogen atom is not H;
each R
5
is independently C
1
-C
10
alkylene;
aaal is the polynucleotide moiety;
p
1
is H, C
1
-C
6
alkyl or an amine protecting group;
a is an integer from 1 to 200;
each b is independently an integer from 1 to 200;
c is an integer from 1 to 10;
d is an integer from 1 to 50; and
each e is independently an integer from 1 to 200.
Preferably, for polyamide of formula I, at least one R
1
is hydrophilic C
1
-C
10
hydrocarbyl.
Another aspect of the present invention provides a polyamide of the formula:
where
each R
10
is independently H or a carboxylic acid protecting group;
each R
11
is independently H, a protecting group or C
1
-C
10
hydrocarbyl;
q is an integer from 1 to 1,200; and
each Q
1
is independently an amino acid side-chain residue or a derivative thereof, provided at least one Q
1
is an amino acid side-chain residue derivative of the formula:
each of the moiety —Q
3
—X
1
—is an amino acid side-chain residue having —X
1
H functional group;
each X
1
is independently O, S or NP
2
;
L is a linker comprising C
1
-C
6
alkylene with carbonyl groups on both of the terminal groups; and
each of L
1
, L
2
, L
3
, L
4
, P
1
, R, R
1
, R
2
, R
3
, R
4
, R
5
, X, a, b, c, d, and e is independently those described above.
Another embodiment of the present invention provides a polyamide-polynucleotide primer conjugate and method for determining the nucleotide sequence of a target nucleic acid which comprises the steps of:
(a) annealing a polyamide-polynucleotide primer conjugate to the target nucleic acid, wherein the polyamide moiety comprises at least one hydrophilic C
1
-C
10
hydrocarbyl substituent on an amide nitrogen atom;
(b) extending the primer with a nucleic acid polymerase in the presence of nucleoside triphosphate precursors and at least one chain terminating nucleotide, thereby forming conjugated nucleic acid fragments;
(c) separating the conjugated nucleic acid fragments by electrophoresis in a non-sieving matrix; and
(d) determining the nucleotide sequence of the target nucleic acid by the separated nucleic acid fragments.
Preferably, the polyamide-polynucleotide primer conjugate comprises a thioether linkage between a polyamide moiety and a polynucleotide moiety. Preferably, the polyamide-polynucleotide primer conjugate is a polypeptoid-polynucleotide primer conjugate of formula I or polypeptide-polynucleotide primer conjugate of formula II above comprising at least one L
3
where one and only one L
3
is the conjugate moiety of the formula:
where aaal is the polynucleotide moiety, wherein the hydroxy group of the terminal 5′-position of the polynucleotide moiety has been replaced with a thiol group to form the thioether linkage between the polyamide moiety and the polynucleotide moiety.
Another embodiment of the present invention provides a method for producing a polyamide of the formula:
comprising contacting a nucleophilic compound of the formula:
with an &agr;, &bgr;-unsaturated carbonyl of the formula:
under conditions sufficient to produce the polyamide III, where
L
5
is a moiety of the formula:
L
6
is a moiety of the formula:
Y
1
is S or NP
2
;
each P
2
is independently H, C
1
-C
6
alkyl or an amine protecting group;
each R
13
is C
1
-C
6
alkylene;
m is an integer from 1 to 200;
n is an integer from 1 to 200;
x is an integer from 0 to 200;
y is an integer from 0 to 10;
z is an integer from 0 to 50;
s is an integer from 0 to 200;
t is an integer from 0 to 10;
u is an integer from 0 to 50; and
each of L
2
, L
3
, L
4
, P
1
, R, R
1
, R
2
, R
3
, R
4
, X, and e is independently those described a

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