Adaptor-based sequence analysis

• US 6,175,002 B1
• Filed: 01/05/1999
• Issued: 01/16/2001
• Est. Priority Date: 04/15/1997
• Status: Expired due to Term

• Current Assignee: Solexa Incorporated (Illumina Incorporated)
• Original Assignee: Lynx Therapeutics, Inc. (Illumina Incorporated)
• Inventors: McCurdy, Sarah N., Brenner, Sydney, Gryaznov, Sergei M., DuBridge, Robert B., Albrecht, Glenn
• Primary Examiner(s): Schwartzman, Robert A.
• Assistant Examiner(s): LARSON, THOMAS GEORGE
• Application Number:
  US09/225,652
• Time in Patent Office: 742 Days
• Field of Search: 435/6, 536/24.2, 536/25.3, 536/23.1
• US Class Current: 536/23.1
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/525 Phosphatase
• CPC Class Codes: C12Q 2525/15 incorporating a consensus or conserved sequence
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor

Abstract

An improvement in adaptor-based sequence analysis is provided that addresses the problems created by self-ligation of target polynucleotides that have complementary ends. The improvement includes preparation of target polynucleotides with dephosphorylated 5′ strands and the use of adaptors having a 3′ blocking group. In a preferred embodiment, adaptors are ligated to target polynucleotides by a single strand, 3′ blocking groups are removed, the adjacent 5′ hydroxyl of the target polynucleotide is phosphorylated, and the ligation of the adaptor is completed by treatment with a ligase.

Claims

1. A double stranded nucleic acid adaptor having a 3′

blocked protuding strand, of the formula;

$\begin{array}{c}\text{\hspace{1em}<br><br>}<br><br>{p<br><br>\left(N\right)}_q-3^{\mathrm{\prime <br><br>}}\\ 3^{\mathrm{\prime <br><br>}}-{z<br><br>\left(N\right)<br><br>\text{\hspace{1em}<br><br>}}_n<br><br>{\left(N^{\mathrm{\prime <br><br>}}\right)}_r-5^{\mathrm{\prime <br><br>}}<br><br>\text{\hspace{1em}<br><br>}\end{array}$

where

N and N′

are complementary nucleotides, p is a phosphate group, z is a 3′

blocking group, n is an integer between 2 and 5, inclusive, q is an integer greater than or equal to 8, r is an integer greater than or equal to 8 which may be the same or different from q, and the 3′

blocked protruding strand is 2 to 6 nucleotides long.

2. The adaptor of claim 1 wherein z is a phosphate group, q is in the range of between 12 and 50, and r is in the range of between 12 and 50.

3. The adaptor of claim 1, where the 3′

blocked protruding strand is 4 nucleotides long.

4. A double stranded nucleic acid adaptor having a 5′

phosphorylated protruding strand, of the formula;

$\begin{array}{c}{5}^{\mathrm{\prime <br><br>}}-p<br><br>\left(N\right)<br><br>{\hspace{0.17em}<br><br>}_n<br><br>\text{\hspace{1em}<br><br>}<br><br>{\left(N\right)}_q-3^{\mathrm{\prime <br><br>}}\\ {z<br><br>\text{\hspace{1em}<br><br>}\left(N^{\mathrm{\prime <br><br>}}\right)}_r-5^{\mathrm{\prime <br><br>}}\end{array}$

where

N and N′

are complementary nucleotides, p is a phosphate group, z is a 3′

blocking group, n is an integer between 2 and 5, inclusive, q is an integer greater than or equal to 8, r is an integer greater than or equal to 8 which may be the same or different frog q, and the 5′

protruding strand is from 2 to 6 nucleotides long.

5. A composition of matter comprising a microparticle having a uniform population of substantially identical double stranded polynucleotides attached, each polynucleotide of the uniform population having a 5′

-dephosphorylated protruding strand at an end distal to the microparticle.

6. The composition of claim 5 wherein said protruding strand is between 2 and 5 nucleotides in length.

7. The composition of claim 6 wherein said uniform population of substantially identical polynucleotides is a population of substantially identical cDNAs.

8. The composition of claim 7 wherein said microparticle is selected from the group consisting of CPG microparticles, GMA microparticles, and polystyrene microparticles.