# US 2016/0355879 A1 · Amplification and analysis of whole genome and whole transcriptome libraries generated by a dna polymerization process

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- Number: US 2016/0355879 A1
- Document ID: US20160355879A1
- Status: Published application
- Kind: A1
- Application number: 15/216,783
- Filed: 2016-07-22
- Published: 2016-12-08
- Earliest claimed: 2003-03-07
- Assignees: Rubicon Genomics, Inc.
- Applicants: Rubicon Genomics, Inc.
- Inventors: Emmanuel KAMBEROV; Tong SUN; Eric BRUENING; Jonathon H. PINTER; Irina SLEPTSOVA; Takao KURIHARA; Vladimir L. MAKAROV
- Web page: https://patentgrep.com/patent/US20160355879A1

## CPC

- C12Q 1/6853: using modified primers or templates
- C12N 15/10: Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34)
- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention regards a variety of methods and compositions for whole genome amplification and whole transcriptome amplification. In a particular aspect of the present invention, there is a method of amplifying a genome comprising a library generation step followed by a library amplification step. In specific embodiments, the library generating step utilizes specific primer mixtures and a DNA polymerase, wherein the specific primer mixtures are designed to eliminate ability to self-hybridize and/or hybridize to other primers within a mixture but efficiently and frequently prime nucleic acid templates.

## Claims

### Claim 1

A method of preparing a plurality of nucleic acid molecules having known constant region at each end, comprising:
a) obtaining a sample comprising nucleic acid molecules;
b) subjecting said nucleic acid molecules to a population of primers to form a nucleic acid molecule/primer mixture, wherein the primers of the population have a nucleotide sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the population, wherein the primers are composed of a constant region and a variable region, the constant region being positioned 5′ to the variable region, wherein primers in the population comprise non-complementary and non-self-complementary nucleotides selected from guanines and adenines; cytosines and thymidines/uridines; adenines and cytosines; or guanines and thymidines/uridines in a relative proportion effective to render the polynucleotides substantially incapable of at least one of the following:
self-hybridization;
self-priming;
hybridization to another polynucleotide in the plurality; or
initiation of a polymerization reaction in the plurality; and
c) subjecting said nucleic acid molecule/primer mixture to a polymerase under conditions to generate the plurality of molecules including the known constant region at each end.

### Claim 2 (depends on 1)

The method of claim 1, wherein said nucleic acid molecules comprise single stranded nucleic acid molecules.

### Claim 3 (depends on 2)

The method of claim 2, wherein said single stranded nucleic acid molecules comprise DNA, RNA, or DNA-RNA chimeras.

### Claim 4 (depends on 1)

The method of claim 1, wherein said nucleic acid molecules are double stranded nucleic acid molecules.

### Claim 5 (depends on 4)

The method of claim 4, wherein said double stranded nucleic acid molecules are DNA, RNA, or DNA-RNA chimeras.

### Claim 6 (depends on 1)

The method of claim 1, wherein said nucleic acid molecules are a mixture of single stranded and double stranded nucleic acid molecules.

### Claim 7 (depends on 6)

The method of claim 6, wherein said single stranded molecules are RNA and said double stranded molecules are DNA.

### Claim 8 (depends on 1)

The method of claim 1, wherein the method further comprises a step d) of amplifying the plurality of the molecules to produce amplified molecules.

### Claim 9 (depends on 8)

The method of claim 8, wherein said amplifying step is carried out using a polymerase chain reaction.

### Claim at position 10

-13. (canceled)

### Claim 14 (depends on 1)

The method of claim 1, wherein said constant region is 6 to 100 nucleotides in length.

### Claim 15 (depends on 1)

The method of claim 1, wherein said variable region is 4 nucleotides to 20 nucleotides in length.

### Claim 16 (depends on 1)

The method of claim 1, wherein the primers of the population further comprise 0 to 3 random bases at its distal 3′ end.

### Claim 17 (depends on 1)

The method of claim 1, wherein the constant region and the variable region are each comprised of guanines and thymidines and wherein the primers of the population further comprise 0, 1, 2, or 3 random bases at their 3′ ends.

### Claim at position 15

-30. (canceled)

### Claim 31 (depends on 8)

The method of claim 8, wherein said method further comprises the steps of:
modifying the amplified molecules to incorporate modified nucleotide bases, thereby producing labeled molecules, said amplified molecules further defined as single stranded DNA, double stranded DNA, or a mixture thereof;
generating single stranded molecules from the labeled molecules, said single stranded molecules capable of hybridizing to complementary sequences arrayed in known locations on a substrate; and
analyzing at least one hybridization signal.

### Claim at position 17

-37. (canceled)

### Claim 38 (depends on 8)

The method of claim 8, wherein a tag is incorporated on the ends of the amplified molecules and wherein said constant region is penultimate to the tags on each end of the amplified molecules.

### Claim at position 19

-47. (canceled)

### Claim 48 (depends on 1)

The method of claim 1, wherein the polymerase is a thermostable polymerase.

### Claim 49

(canceled)

### Claim 50 (depends on 1)

The method of claim 1, wherein the nucleic acid molecule/primer mixture is subjected to polymerase chain reaction having polymerization steps comprising two or more DNA denaturation steps.

### Claim 51 (depends on 50)

The method of claim 50, wherein step c) is further defined as comprising:
(1) a first series of polymerase chain reaction steps comprising subjecting the nucleic acid molecule/primer mixture to a thermostable polymerase in the presence of the primers under conditions having two or more cycling DNA denaturation steps; and
(2) a second series of polymerase chain reaction steps using primers that comprise the constant region sequence.

### Claim 52 (depends on 1)

The method of claim 1, wherein said nucleic acid sequence is further defined as rendering the polynucleotides substantially incapable of the following:
self-hybridization;
self-priming;
hybridization to another polynucleotide in the plurality; and
initiation of a polymerization reaction in the plurality.

### Claim 53 (depends on 1)

The method of claim 1, wherein at least 75% of a sequence of a primer in the population is comprised of two non-complementary and non-self-complementary nucleotides.

### Claim 54 (depends on 53)

The method of claim 53, wherein at least 80% of a sequence of a primer in the population is comprised of two non-complementary and non-self-complementary nucleotides.

### Claim 55 (depends on 54)

The method of claim 54, wherein at least 85% of a sequence of a primer in the population is comprised of two non-complementary and non-self-complementary nucleotides.

### Claim 56 (depends on 55)

The method of claim 55, wherein at least 90% of a sequence of a primer in the population is comprised of two non-complementary and non-self-complementary nucleotides.

### Claim 57 (depends on 56)

The method of claim 56, wherein at least 95% of a sequence of a primer in the population is comprised of two non-complementary and non-self-complementary nucleotides.

### Claim 58 (depends on 57)

The method of claim 57, wherein at least 97% of a sequence of a primer in the population is comprised of two non-complementary and non-self-complementary nucleotides.

### Claim 59 (depends on 58)

The population of claim 58, wherein 100% of a sequence of a primer in the population is comprised of two non-complementary and non-self-complementary nucleotides.