Methods for preparing nucleic acid samples

A method of synthesizing a preparation of nucleic acid molecules, the method comprising the steps of:

• (a) utilizing an RNA template to enzymatically synthesize a first DNA molecule that is complementary to at least 50 contiguous bases of the RNA template, wherein:
• (i) the first DNA molecule is synthesized using reverse transcriptase;
• (ii) the synthesis of the first DNA molecule is primed using a first primer mixture comprising a multiplicity of first primers, wherein each of the first primers comprises a random sequence portion and a defined sequence portion located 5′ to the random sequence portion, wherein the defined sequence portion comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:3;
• (b) hydrolyzing the template RNA and removing substantially all of the first primer mixture after synthesis of the first DNA molecule;
• (c) utilizing the first DNA molecule as a template to enzymatically synthesize a second DNA molecule, thereby forming a double-stranded DNA molecule wherein the first DNA molecule is hybridized to the second DNA molecule, wherein:
• (i) the second DNA molecule is synthesized using the Klenow fragment of DNA polymerase I;
• (ii) the synthesis of the second DNA molecule is primed using a second primer mixture comprising a multiplicity of second primer molecules, wherein each second primer molecule comprises a random sequence portion and a defined sequence portion, wherein the sequence of the defined sequence portion of each second primer molecule is different from the sequence of the defined sequence portion of each first primer molecule, and wherein the second primer defined sequence portion comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:3;
• (d) removing substantially all of the second primer mixture after synthesis of the second DNA molecule;
• (e) amplifying the double-stranded DNA molecule using a polymerase chain reaction comprising from 5 to 15 amplification cycles, wherein the polymerase chain reaction is primed with a first PCR primer population and a second PCR primer population, wherein the first PCR primer population consists essentially of primer molecules consisting of the sequence set forth in SEQ ID NO:5, and the second PCR primer population consists essentially of primer molecules consisting of the sequence set forth in SEQ ID NO:6;
• (f) removing substantially all of the PCR primer mixture after amplification of the double-stranded DNA molecule;
• (g) utilizing the first or second DNA molecule of the amplified, double-stranded, DNA molecule as a template to synthesize, using an RNA polymerase molecule, a first RNA molecule that is complementary to either the first DNA molecule or to the second DNA molecule;
• (h) purifying the first RNA molecule to remove substantially all nucleic acid molecules less than 100 bases long;
• (i) utilizing the first RNA molecule as a template to enzymatically synthesize a third DNA molecule that is complementary to the first RNA molecule, wherein the third DNA molecule is synthesized using reverse transcriptase and the synthesis of the third DNA molecule is primed using a population of random primers wherein substantially all of the random primers consist of 9 bases;
• and
• (j) joining Cy dye molecules to the third DNA molecule by aminoallyl linkages.

A DNA sample prepared by a method comprising the steps of:

• (a) utilizing an RNA template to enzymatically synthesize a first DNA molecule that is complementary to at least 50 contiguous bases of said RNA template;
• (b) utilizing the first DNA molecule as a template to enzymatically synthesize a second DNA molecule thereby forming a double-stranded DNA molecule wherein the first DNA molecule is hybridized to the second DNA molecule;
• (c) utilizing the first or second DNA molecule of the double-stranded DNA molecule as a template to enzymatically synthesize a first RNA molecule that is complementary to either the first DNA molecule or to the second DNA molecule;
• and
• (d) utilizing the first RNA molecule as a template to enzymatically synthesize a third DNA molecule that is complementary to the first RNA molecule.

A method for hybridizing a processed DNA sample to a population of immobilized nucleic acid molecules, the method comprising the step of hybridizing a processed DNA sample to a population of immobilized nucleic acid molecules, wherein the processed DNA sample is prepared by a method comprising the steps of:

• (a) utilizing an RNA template to enzymatically synthesize a first DNA molecule that is complementary to at least 50 contiguous bases of said RNA template;
• (b) utilizing the first DNA molecule as a template to enzymatically synthesize a second DNA molecule, thereby forming a double-stranded DNA molecule wherein the first DNA molecule is hybridized to the second DNA molecule;
• (c) utilizing the first or second DNA molecule of the double-stranded DNA molecule as a template to enzymatically synthesize a first RNA molecule that is complementary to either the first DNA molecule or to the second DNA molecule;
• and
• (d) utilizing the first RNA molecule as a template to enzymatically synthesize a third DNA molecule that is complementary to the first RNA molecule.