Highly multiplex pcr methods and compositions

Abstract

The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.

What is claimed is: 
     
         1 . A method of selecting test primers from a library of candidate primers, the method comprising:
 (a) calculating on a computer an undesirability score for most or all of the possible combinations of two candidate primers from the library, wherein each undesirability score is based at least in part on the likelihood of dimer formation between the two candidate primers; and   (b) removing the candidate primer with the highest undesirability score from the library of candidate primers, thereby selecting a library of test primers.   
     
     
         2 . The method of  claim 1 , further comprising:
 (c) if the candidate primer removed in step (b) is a member of a primer pair, then removing the other member of the primer pair from the library of candidate primers; and   (d) optionally repeating steps (b) and (c), thereby selecting a library of test primers.   
     
     
         3 . The method of  claim 1 , wherein the undesirability scores are based at least in part on one or more parameters selected from the group consisting of heterozygosity rate of the target locus, disease prevalence associated with a polymorphism or mutation at the target locus, disease penetrance associated with a polymorphism or mutation at the target locus, specificity of the candidate primer for the target locus, size of the candidate primer, melting temperature of the target amplicon, guanine-cytosine (GC) content of the target amplicon, amplification efficiency of the target amplicon, and size of the target amplicon. 
     
     
         4 . A method of selecting test primers from a library of candidate primers, the method comprising:
 (a) calculating on a computer an undesirability score for most or all of the possible combinations of two candidate primers from the library, wherein each undesirability score is based at least in part on the likelihood of dimer formation between the two candidate primers; and   (b) removing from the library of candidate primers the candidate primer that is part of the greatest number of combinations of two candidate primers with an undesirability score above a first minimum threshold, thereby selecting a library of test primers.   
     
     
         5 . The method of  claim 4 , further comprising:
 (c) if the candidate primer removed in step (b) is a member of a primer pair, then removing the other member of the primer pair from the library of candidate primers; and   (d) optionally repeating steps (b) and (c), thereby selecting a library of test primers.   
     
     
         6 . The method of  claim 5 , comprising further reducing the number of candidate primers remaining in the library by decreasing the first minimum threshold used in step (b) to a lower second minimum threshold and repeating steps (b) and (c) until the undesirability scores for the candidate primer combinations remaining in the library are all equal to or below the second minimum threshold, or until the number of candidate primers remaining in the library is reduced to a desired number. 
     
     
         7 . The method of  claim 5 , comprising increasing the first minimum threshold used in step (b) to a higher second minimum threshold and repeating steps (b) and (c) until the undesirability scores for the candidate primer combinations remaining in the library are all equal to or below the second minimum threshold, or until the number of candidate primers remaining in the library is reduced to a desired number. 
     
     
         8 . The method of  claim 4 , wherein a candidate primer is selected out of a group of two or more candidate primers with equal undesirability scores for removal from the library of candidate primers based on one or more other parameters. 
     
     
         9 . The method of  claim 4 , wherein the undesirability scores are based at least in part on one or more parameters selected from the group consisting of heterozygosity rate of the target locus, disease prevalence associated with a polymorphism or mutation at the target locus, disease penetrance associated with a polymorphism or mutation at the target locus, specificity of the candidate primer for the target locus, size of the candidate primer, melting temperature of the target amplicon, GC content of the target amplicon, amplification efficiency of the target amplicon, and size of the target amplicon. 
     
     
         10 . The method of  claim 9 , wherein the undesirability scores are based at least in part on one or more parameters selected from the group consisting of heterozygosity rate of the target locus, specificity of the candidate primer for the target locus, size of the candidate primer, melting temperature of the target amplicon, GC content of the target amplicon, amplification efficiency of the target amplicon, and size of the target amplicon; and wherein the test primers are used to simultaneously amplify at least 1,000 target loci in a sample comprising maternal DNA from the pregnant mother of a fetus and fetal DNA to determine the presence or absence of a fetal chromosome abnormality. 
     
     
         11 . The method of  claim 10 , comprising ligating a universal primer binding site to the DNA molecules in the sample; amplifying the ligated DNA molecules using at least 1,000 specific primers and a universal primer to produce a first set of amplified products; and amplifying the first set of amplified products using at least 1,000 pairs of specific primers to produce a second set of amplified products. 
     
     
         12 . The method of  claim 9 , wherein the undesirability scores are based at least in part on one or more parameters selected from the group consisting of heterozygosity rate of the target locus, specificity of the candidate primer for the target locus, size of the candidate primer, melting temperature of the target amplicon, GC content of the target amplicon, amplification efficiency of the target amplicon, and size of the target amplicon; and wherein the test primers are used to simultaneously amplify at least 1,000 target loci in sample comprises DNA from an alleged father of a fetus and to simultaneously amplify the target loci in a sample comprising maternal DNA from the pregnant mother of a fetus and fetal DNA to establish whether the alleged father is the biological father of the fetus. 
     
     
         13 . The method of  claim 9 , comprising using the test primers to simultaneously amplify at least 1,000 target loci in a control nucleic acid sample to produce a first set of target amplicons and to simultaneously amplify the target loci in a test nucleic acid sample to produce a second set of target amplicons; and comparing the first and second sets of target amplicons to determine whether a target locus is present in one sample but absent in the other, or whether a target locus is present at different levels in the control sample and the test sample. 
     
     
         14 . The method of  claim 9 , comprising using the test primers to simultaneously amplify at least 1,000 target loci in a control sample comprising RNA to produce a first set of target amplicons and to simultaneously amplify the target loci in a test sample comprising RNA to produce a second set of target amplicons; and comparing the first and second sets of target amplicons to determine the presence or absence of a difference in the RNA expression levels between the control sample and the test sample. 
     
     
         15 . The method of  claim 4 , further comprising, prior to step (b), removing a primer pair from the library that produces a target amplicon that overlaps with a target amplicon produced by another primer pair. 
     
     
         16 . The method of  claim 4 , wherein a range of GC content among the different test primers in the library is less than 30%, wherein a range of melting temperatures among the different test primers in the library is less than 20° C., and wherein a range of the length among the different target amplicons is less than 50 nucleotides. 
     
     
         17 . The method of  claim 4 , wherein the test primers comprise a 5′ region that is specific for a target locus, an internal region that is not specific for the target locus and forms loop structure, and a 3′ region that is specific for a different portion of the same target locus 
     
     
         18 . The method of  claim 4 , further comprising:
 contacting a nucleic acid sample comprising target loci with the test primers to produce a reaction mixture; and   subjecting the reaction mixture to primer extension reaction conditions to produce amplified products comprising target amplicons.   
     
     
         19 . The method of  claim 18 , wherein the test primers simultaneously amplify at least 1,000 different target loci. 
     
     
         20 . The method of  claim 19 , wherein the test primers simultaneously amplify at least 10,000 different target loci. 
     
     
         21 . The method of  claim 18 , wherein at least 95% of the amplified products are target amplicons. 
     
     
         22 . The method of  claim 18 , wherein less than 10% of the amplified products are primer dimers. 
     
     
         23 . The method of  claim 18 , wherein at least 80% of the target loci are amplified. 
     
     
         24 . The method of  claim 18 , wherein the sample comprises maternal DNA from the pregnant mother of a fetus and fetal DNA, and wherein the method further comprises:
 sequencing the amplified products to obtain a plurality of sequence tags aligning to the target loci; wherein the sequence tags are of sufficient length to be assigned to a specific target locus;   assigning on a computer the plurality of sequence tags to their corresponding target loci;   determining on a computer a number of sequence tags aligning to the target loci of a first chromosome and a number of sequence tags aligning to the target loci of a second chromosome; and   comparing the number for the first chromosome to the number for the second chromosome to determine the presence or absence of an abnormal distribution of the first chromosome.   
     
     
         25 . The method of  claim 24 , wherein the abnormal distribution of the first chromosome is indicative of a fetal aneuploidy, Down syndrome, Edwards syndrome, Patau syndrome, Turner syndrome, Klinefelter's syndrome, Prader-Willi syndrome, DiGeorge syndrome, Angelman syndrome, or Beckwith-Wiedemann syndrome. 
     
     
         26 . The method of  claim 4 , comprising selecting a library of at least 500 different test primers. 
     
     
         27 . The method of  claim 26 , comprising selecting a library of at least 5,000 different test primers. 
     
     
         28 . The method of  claim 27 , comprising selecting a library of at least 20,000 different test primers. 
     
     
         29 . A library of test primers selected using the method of  claim 1 . 
     
     
         30 . A library of test primers selected using the method of  claim 4 .