Polymerase Chain Reaction (PCR) enables specific amplification of as little as a single copy of a target nucleic acid sequence. High sensitivity and specificity of PCR proved to be of great value in diagnostics, forensics and other applications where a small amount of target nucleic acid must be detected. Unfortunately, the same high sensitivity of PCR assays makes them vulnerable to contamination and false positive results. In forensics and disease screening (such as HIV testing), false positive results can have devastating consequences.
The most common source of false positive results is “carryover contamination”, where a PCR product (amplicon) from a prior assay contaminates subsequent PCR assays. The contaminant may be transmitted by a technician, an instrument or even via aerosol. In a “negative” sample, where the target nucleic acid is absent, the contaminant creates a false positive result. In a “positive” sample, where the target nucleic acid is present, the contaminant is co-amplified with the true target. Such co-amplification may distort a result of a quantitative assay, where exact amount of the true target must be determined.
A popular and effective way of preventing carryover contamination involves the use of uracil DNA glycosylases, specifically UNG (EC 3.2.2.3). These enzymes recognize uracils present in single-stranded or double-stranded DNA and cleave the N-glycosidic bond between the uracil base and the deoxyribose, leaving an abasic site. See e.g. U.S. Pat. No. 6,713,294. Uracil-DNA glycosylases, abbreviated as “UDG” or “UNG” include mitochondrial UNG1, nuclear UNG2, SMUG1 (single strand-selective uracil-DNA glycosylase), TDG (TU mismatch DNA glycosylase), MBD4 (uracil-DNA glycosylase with a methyl binding domain) and other eukaryotic and prokaryotic enzymes (See Krokan H. E. et al. “Uracil in DNA—occurrence, consequences and repair”, Oncogene (2002) 21:8935-9232).
Uracil-DNA glycosylases are DNA repair enzymes that prevent among others, G to A transition mutations caused by deamination of cytosine into uracil. If cytosine (C) is deaminated into a uracil (U) and the DNA undergoes replication, an A would be incorporated opposite the U, where G was previously located opposite the C. If the uracil base is excised by the glycosylase prior to replication, the abasic site is repaired by short-patch or long-patch DNA repair pathway, involving endonuclease and DNA polymerase activities. In addition to DNA damage repair, DNA glycosylase activity plays a role in somatic mutation, including immunoglobulin class switch and somatic hypermutation during antibody affinity maturation. See Bransteitter R. et al. “First AID (Activation-induced cytidine deaminase) is needed to produce high affinity isotype-switched antibodies”, J. Bio. Chem. (2007) 281:16833-16836.
Preparation of uracil-N-DNA glycosylase (UNG) optimized for the control of carryover contamination in amplification reactions has been disclosed for example, in the U.S. Pat. No. 6,187,575. The use of UNG to prevent carryover contamination has also been described. See Longo et al. “Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reaction” (1990) Gene, 93:125-128. State of the art method of controlling carryover contamination using UNG is described in U.S. Pat. Nos. 6,287,823 and 6,518,026 and U.S. Pub. No. 2003/0077637.
Generally, the method involves two steps. First, the PCR assays must include dUTP, so that the amplicons, which are potential carryover contaminants, contain uracil. The method involves substituting dUTP for some or all of the dTTP in the amplification reaction. Alternatively (or in addition), one or more uracils may be incorporated into the amplification primers. It should be noted, however, that if a uracil in the primer is too close to the 5′-end, the method is less efficient at preventing subsequent amplification. The use of dUTP does not interfere with PCR assays. After a uracil-containing amplicon is generated, it can be detected and analyzed by standard methods despite the presence of uracil in place of thymine.
Next, uracil-N-DNA glycosylase is added to a subsequent PCR. Conveniently, UNG is active in a standard reaction mixture that contains all the components of PCR. This enables adding UNG to assembled PCR reactions or even to the PCR master mix. Before the start of thermal cycling, the reaction mixture is incubated at a temperature optimal for the UNG activity within the context of the PCR master mix (about 50° C.) or within the temperature range where UNG is active. If a uracil-containing contaminant from a prior reaction is present, UNG will cleave off the uracil, leaving an abasic site. DNA with abasic sites is known to be labile at high temperature under high pH conditions. When the thermal cycling begins, such DNA is degraded. The high temperature also inactivates the UNG enzyme, allowing to generate new DNA amplicons containing uracil.
After treatment with UNG, abasic DNA must be efficiently cleaved at the abasic sites. Unless it is cleaved, abasic DNA becomes a template for the polymerase in subsequent amplification. For example, Taq DNA polymerase is known to bypass abasic sites by incorporating an adenosine opposite the missing base. A single bypass by the polymerase generates a perfect template for subsequent amplification (see Sikorsky, J. A. et al., “DNA damage reduces Taq polymerase fidelity and PCR amplification efficiency”, Biochem. Biophys. Res. Commun. (2007) 355:431-437 or Kobayashi, A. et al. “Novel PCR-mediated mutagenesis employing DNA containing a natural abasic site as a template and translesional Taq DNA polymerase”, J. Biotech. (2005) 116:227-232). Therefore the ability to efficiently cleave DNA at the abasic sites is essential for the overall success of the UNG-based method of preventing carryover contamination.
USPTO Backgrounds
USPTO Backgrounds