Null A P, Hannis J C, Muddiman D C
Department of Chemistry, Virginia Commonwealth University, Richmond 23284, USA.
Analyst. 2000 Apr;125(4):619-26. doi: 10.1039/a908022h.
Electrospray ionization mass spectrometry (ESI-MS) has been utilized to obtain accurate mass measurements of intact PCR products; however, single-stranded PCR products are necessary to detect sequence modifications such as base substitutions, additions or deletions. The locations of these modifications can subsequently be determined using additional stages of mass spectrometry. The recombinant enzyme lambda exonuclease selectively digests one strand of a DNA duplex from a 5' phosphorylated end leaving the complementary strand intact. Using this rapid enzymatic step, we were able to produce single-stranded PCR products by digestion of an intact PCR product derived from the Human Tyrosine Hydroxylase (HUMTHO1) gene, which contains a tetrameric repeating motif. The non-template directed 3' adenylation common when using Taq polymerase resulted in three distinct species (blunt-ended, mono-adenylated and di-adenylated), which added complexity to the spectrum of the double-stranded product. The data from the single-stranded products shows that one strand is preferentially adenylated over the other, which cannot be determined from the mass spectrum of the double-stranded PCR product alone. The ESI-FTICR (Fourier transform ion cyclotron resonance) mass spectra of the lambda exonuclease treated PCR products exhibited less than expected signal-to-noise (S/N) ratios. This is attributed to inaccurate concentration calculations due to remaining double-stranded PCR product amplified with unphosphorylated primers, and to matrix effects contributed by the lambda exonuclease reaction buffer. To further test this hypothesis, we investigated and determined the limit of detection to be 0.27 microM using standard curve statistics for single acquisitions of a synthetic 75-mer. The concentrations of the noncoding and coding strands produced by lambda exonuclease digestion were calculated to be 0.29 and 0.37 microM, respectively, taking into account the presence of double-stranded product. The products were electrosprayed from concentrations at the limit of detection requiring the averaging of 5-10 acquisitions to produce a sufficient S/N ratio, indicating that product concentration, base composition and matrix effects play a combined, significant role in detection of lambda exonuclease treated PCR products. Although additional work will be required to further exploit this strategy, lambda exonuclease clearly provides mass spectrometrists with a method to generate single-stranded PCR products.
电喷雾电离质谱（ESI-MS）已被用于获得完整PCR产物的精确质量测量；然而，检测诸如碱基替换、添加或缺失等序列修饰需要单链PCR产物。这些修饰的位置随后可使用质谱的其他阶段来确定。重组酶λ外切核酸酶从5'磷酸化末端选择性地消化DNA双链体的一条链，使互补链保持完整。通过这个快速的酶促步骤，我们能够通过消化源自人酪氨酸羟化酶（HUMTHO1）基因的完整PCR产物来产生单链PCR产物，该基因包含一个四聚体重复基序。使用Taq聚合酶时常见的非模板导向3'腺苷化导致了三种不同的产物（平端、单腺苷化和双腺苷化），这增加了双链产物谱的复杂性。单链产物的数据表明，一条链比另一条链更优先被腺苷化，这仅从双链PCR产物的质谱中无法确定。λ外切核酸酶处理的PCR产物的ESI-FTICR（傅里叶变换离子回旋共振）质谱显示出低于预期的信噪比（S/N）。这归因于用未磷酸化引物扩增的剩余双链PCR产物导致的浓度计算不准确，以及λ外切核酸酶反应缓冲液造成的基质效应。为了进一步验证这一假设，我们使用合成75聚体单次采集的标准曲线统计方法，研究并确定检测限为0.27微摩尔。考虑到双链产物的存在，计算出λ外切核酸酶消化产生的非编码链和编码链的浓度分别为0.29和0.37微摩尔。产物从检测限浓度进行电喷雾，需要对5-10次采集进行平均以产生足够的信噪比，这表明产物浓度、碱基组成和基质效应在检测λ外切核酸酶处理的PCR产物中共同发挥着重要作用。尽管需要进一步开展工作来进一步开发这一策略，但λ外切核酸酶显然为质谱分析人员提供了一种生成单链PCR产物的方法。
