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肽核酸(PNAs):新一代遗传学和细胞遗传学分析探针

(2007-01-11 17:43:49)
The peptide nucleic acids (PNAs): a new generation of probes for genetic and cytogenetic analyses
肽核酸(PNAs):新一代遗传学和细胞遗传学分析探针

Abstract:Peptide nucleic acids (PNAs) are synthetic homologs of nucleic acids in which the phosphate–sugar polynucleotide backbone is replaced by a flexible pseudo-peptide polymer to which the nucleobases are linked.
摘要:肽核酸(PNAs)是一类人工合成的核酸同系物,它的磷酸-糖多核苷酸骨架被与核苷酸碱基连接的易弯曲假肽核酸聚合物所代替。
This structure gives PNAs the capacity to hybridize with high affinity and specificity to complementary sequences of DNA and RNA, and also confers remarkable resistance to DNAses and proteinases.
这种结构不但使得PNAs在与DNA和RNA互补序列杂交时表现出强的亲和力和高特异性,并且对脱氧核糖核酸酶与蛋白酶有很强的抵抗能力。
The unique physico-chemical characteristics of PNAs have led to the development of a wide range of biological assays. Several exciting new applications of PNA technology have been published recently in genetics and cytogenetics.
PNAs独特的理化性质拓宽了生物分析的应用范围。在遗传学和细胞遗传学方面,最近已陆续报道了几个有关PNA技术令人兴奋的新应用。
Also, PNA-based hybridization technology is developing rapidly within the field of in situ fluorescence hybridization, pointing out the great potential of PNA probes for chromosomal investigations.
而且,以PNA为基础的杂交技术在原位荧光杂交领域的迅速发展,也预示着PNA探针在染色体研究方面的巨大潜能。
Introducetion:In human cytogenetics, the advent of fluorescence molecular techniques has brought forth new procedures for chromosome investigation and diagnosis. The fluorescence in situ hybridization (FISH) technique has established itself as a powerful improvement over conventional cytogenetic techniques and is now a well-accepted procedure for chromosome analysis and aneuploidy screening.
前言:在人类细胞遗传学中,荧光分子技术的出现带来了一些染色体研究和诊断的新方法。相对于传统的细胞遗传学方法,原位杂交技术(FISH)具有很大的改进,已成为一种公认的染色体分析和非整倍体筛选(aneuploidy screening)技术。
Because of its performance, its easy implementation and the commercial availability of numerous DNA probes, FISH has quickly found a large spectrum of applications in chromosomal research and diagnosis.
FISH技术不但检测灵敏、易于操作,而且已有许多DNA探针可商业化提供,因此很快便在染色体研究和诊断领域被广泛应用。
The primed in situ (PRINS) method, based on PCR principles, has offered an interesting alternative approach for the identification of human chromosomes. Although this technique has proven its efficiency on several types of cells, its current utilization is limited to the detection of repeated DNA sequences and requires the use of sequential reactions.
最初在PCR(聚合酶链式反应)原理上建立的原位检测技术——寡核苷酸引物原位DNA检测(PRINS)方法,为人类染色体的鉴定提供了一个有趣的可供选择(alternative)的手段。虽然这种技术在某些类型细胞上的应用十分奏效,但是它现在主要被用于探测重复DNA序列,而且需要使用连续的反应。
Peptide nucleic acids (PNAs) constitute an attractive new class of probes, recently introduced in cytogenetics. The particular nature and the unique properties of these molecules make their use a promising procedure for in situ hybridization assays. The PNA technology is already used as an essential tool in a wide range of research and diagnostic molecular protocols.
作为一类让人感兴趣的新型探针,肽核酸最近已被引入到细胞遗传学的研究中。这些分子特殊的性质与独有的属性为原位杂交分析提供了一个很有前景的方法。在很多研究与分子诊断领域,PNA技术已成为一项基本研究工具。
During the last years, new applications of PNA have emerged in genetics and cytogenetics, enabling development of robust and simplified procedures for genome mapping, antigene therapy, mutation detection or aneuploidy screening.
    近几年来,PNA在遗传学和细胞遗传学领域的新应用,促进了遗传图谱、抗基因治疗、突变检测及非整倍体筛选方面的高效、简便技术的发展。
PNAs are synthetic DNA analogs in which the phosphodiester backbone is replaced by repetitive units of N-(2-aminoethyl) glycine to which the purine and pyrimidine bases are attached via a methyl carbonyl linker.The PNA molecules can routinely be labeled with biotin or various fluorophores.
PNA是人工合成的DNA同类物,它的磷酸二酯骨架被重复的N-(2-氨乙基)-甘氨酸单位所取代,而嘌呤和嘧啶系通过甲基羰基接枝。PNA分子可被生物素和各种荧光团常规标记。
The unique chemical makeup provides PNA with unique hybridization characteristics. Unlike DNA and RNA, the PNA backbone is not charged. Consequently, there is no electrostatic repulsion when PNA hybridizes to its target nucleic acid sequence, giving a higher stability to the PNA–DNA or PNA–RNA duplexes than the natural homo- or heteroduplexes.
PNA独特的化学组成使其具有特异的杂交性能。与DNA或RNA不同,PNA的骨架不带负电荷。因此,当PNA与靶核酸序列杂交时不存在静电排斥,从而使PNA-DNA和PNA-RNA双链比野生的同源或异源双链具有更好的稳定性。
This greater stability results in higher thermal melting temperature (Tm) values than is observed for DNA–DNA or DNA–RNA duplexes. An additional consequence of the polyamide backbone is that PNAs hybridize virtually independently of the salt concentration. Thus, the Tm of PNA–DNA duplex is barely affected by low ionic strength. This significantly facilitates the hybridization with the PNAs.
这种稳定性使它的热熔化温度值(Tm)比DNA-DNA 和 DNA-RNA 双链要高。关于聚酰胺骨架的另一个推论是: PNA的杂交与溶液盐溶度无关。因此,PNA-DNA 的热熔温度几乎不受弱离子强度的影响,十分有利于PNA的杂交。
The unnatural backbone of PNAs also means that PNAs are particularly resistant to protease and nuclease degradation.Because of this resistance to the enzyme degradation, the lifetime of PNAs is extended both in vivo and in vitro. Also, PNAs are not recognized by polymerases and therefore cannot be directly used as primers or be copied.
PNA的非天然骨架也使得PNA能有效对抗蛋白酶和核酸酶的降解。鉴于这种对酶消化能力的抗性,PNA在体内外的寿命都变长。而且,由于PNA不被聚合酶所识别,所以不能被直接用作引物或是被复制。
PNAs hybridize to complementary DNA or RNA in a sequence dependent manner, according to the Watson–Crick hydrogen bonding scheme. In contrast to DNA, PNA can bind in either parallel or anti-parallel fashion.
依照 Watson-Crick 的氢键结合规则,PNA以序列依赖的方式与互补DNA或互补RNA 杂交。与DNA不同,PNA即可以通过正向平行的方式结合,也可以借助反向平行的方式结合。
These data indicate that the PNA backbone is more flexible than native nucleic acid backbone. PNA probes can bind to either single stranded DNA or RNA, or to double stranded DNA.
这些数据表明,PNA骨架比天然的核酸骨架具有更高的灵活性。PNA探针可与单链RNA、DNA或双链的DNA结合。
Homopyrimidine PNAs, as well as PNAs containing a high proportion of pyrimidine residues, bind to complementary DNA sequences to form highly stable (PNA)2–DNA triplex helices displaying Tm over 72 °C. In these triplexes, one PNA strand hybridizes to DNA through standard Watson–Crick base pairing rules, while the other PNA strand binds to DNA through Hoogsteen hydrogen bonds. The resulting structure is called P-loops.
和PNA一样,同型嘧啶( Homopyrimidine ) PNA含有高比例的嘧啶残基,与互补DNA结合形成的高度稳定的(PNA)2-DNA三联体,其热熔温度超过了72度。在这些三联体中,一条 PNA链是依靠Watson–Crick碱基配对原则与DNA结合,另一条PNA则以Hoogsteen 氢键方式结合在DNA上。由此而来的结构称为 P-环 。
The stability of these triple helixes is so high that homopyrimidine PNA targeted to purine tracts of dsDNA invades the duplex by displacing one of the DNA strands. The efficiency of this strand invasion can be further enhanced by using two homopyrimidine PNA oligomers connected via a flexible linker.
这种三螺旋体的稳定性极高,以至于在同型嘧啶PNA靶向双链DNA嘌呤道时,能取代其中一条DNA链侵入双螺旋。如果使用由一个灵活的连接物结合的双同型嘧啶PNA寡聚体,这种链侵入作用的效率可进一步提高。
Also, PNA–DNA hybridization is significantly more affected by base mismatches than DNA–DNA hybridization. A single mismatch in a mixed PNA–DNA 15-mer duplex decreases the Tm by up to 15 °C, whereas in the corresponding DNA–DNA complex, a single mismatch decreases the Tm by only 11 °C.
值得关注的是, PNA-DNA杂交比DNA-DNA杂交更易受碱基误配的影响。在混合型的15-mer PNA-DNA二联体中,单碱基误配可使它的热熔值降低15度,而在相应的 DNA-DNA 二联体中,一个错误匹配只能使热熔值降低11度。
This high level of discrimination at single base level has indicated that short PNA probes could offer high specificity and has thus allowed the further development of several efficient PNA-based strategies for molecular investigations and diagnosis.
这种在单碱基水平上的高度辨别特性预示,短片段的PNA探针具有很高的灵敏性,从而使一些以PNA为基础的策略能够更好地被用于分子研究与诊断。

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