Amplification and analysis of whole genome and whole transcriptome libraries generated by a DNA polymerization process
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.
1. A kit, comprising:
a container; and
a) a population of primers configured to amplify a whole genome or whole transcriptome present in the container, wherein the population of primers are non-self-complementary and non-complementary to other primers in the population, and comprise in a 5′ to 3′ orientation a constant region and a variable region that are adjacent to each other, wherein the constant region sequence has a known sequence that is constant among a plurality of primers of the population and the variable region sequence is degenerate among the plurality of primers of the population, wherein the sequence of the constant region comprises at least 70% of two types of non-complementary nucleotides selected from the group consisting of adenines and guanines; adenines and cytosines; guanines and thymidines; and cytosines and thymidines, and the sequence of the variable region comprises greater than 50% non-complementary nucleotides selected from the group consisting of adenines and guanines; adenines and cytosines; guanines and thymidines; and cytosines and thymidines;
wherein the constant region is 6-100 nucleotides in length;
b) at least one target-specific primer for targeted amplification;
c) at least one nested primer for further amplifying amplicons generated by the targeted amplification; and
d) a set of instructions for using the kit to detect copy number variation (CNV), genotype single nucleotide polymorphism (SNP), genotype micro-satellite, detect insertion and/or deletion, determine zygosity, or detect mutation(s),
wherein the sequence of the constant and variable regions will not cross-hybridize or self-hybridize under conditions described in the set of instructions.
2. The kit of claim 1 , wherein amplicons generated by the primers in (a)-(c) are at least 100 base pairs long.
3. The kit of claim 1 , wherein the primers in (a)-(c) comprise at least 10 nucleotides.
4. The kit of claim 1 , wherein the primers in (a)-(c) comprise at least one modified nucleotide.
5. The kit of claim 1 , wherein melting temperature of the primers in (a)-(c) is at least 45 degrees Celsius.
6. The kit of claim 1 , wherein the at least one nested primer is complementary to amplicons generated by the targeted amplification.
7. The kit of claim 1 , wherein the at least one target-specific primer is specific to one or more target sequences.
8. The kit of claim 1 , wherein the at least one target-specific primer does not comprise an adapter sequence.
9. The kit of claim 1 , wherein the set of instructions instructs a user to carry out a first series of polymerase chain reaction steps using the population of primers and the at least one target-specific primer followed by a second series of polymerase chain reaction steps using the at least one nested primer.
10. The kit of claim 9 , wherein the set of instructions further instructs the user to sequence amplicons generated at the end of the second series of polymerase chain reaction steps using a sequencer.
11. The kit of claim 1 , wherein the kit further comprises reagents selected from the group consisting of a cell lysis reagent, magnesium salts, a ligase, a polymerase, a reverse transcriptase, betaine and a kinase.
12. The kit of claim 1 , wherein the kit is configured for use with a sample comprising nucleic acid molecules: DNA or RNA.
13. The kit of claim 12 , wherein the sample is selected from the group consisting of blood, serum, plasma, cerebrospinal fluid, cheek scrapings, nipple aspirate, biopsy, semen, urine, feces, hair follicle, saliva, sweat, immunoprecipitated or physically isolated chromatin, a single cell or a plurality of cells.
14. The kit of claim 12 , wherein the kit is configured for obtaining genomic copy number, sequence information, allelic variation information, cancer diagnosis, prenatal diagnosis, paternity information, disease diagnosis, detection, monitoring, or treatment information using the sample.
15. The kit according to claim 1 , wherein the variable region comprises 4 to 20 nucleotides.
16. A kit, comprising:
a) a population of primers configured to amplify a whole genome or whole transcriptome, wherein the population of primers are non-self-complementary and non-complementary to other primers in the population, and comprise in a 5′ to 3′ orientation a constant region and a variable region, wherein the constant region sequence has a known sequence that is constant among a plurality of primers of the population and the variable region sequence is degenerate among the plurality of primers of the population, wherein the primers of the population have sequences that are 70% or more comprised of two non-complementary and non-self-complementary nucleotides;
b) at least one target-specific primer for targeted amplification;
c) at least one nested primer for further amplifying amplicons generated by the targeted amplification; and
d) a set of instructions for using the kit to detect copy number variation (CNV), genotype single nucleotide polymorphism (SNP), genotype micro-satellite, detect insertion and/or deletion, determine zygosity, or detect mutation(s),
wherein the sequence of the constant and variable regions will not cross-hybridize or self-hybridize under conditions described in the set of instructions.