Introduction

Zip nucleic acids (ZNA)는 cationic spermine unit과 결합시킨 oligonucleotide로 음전하-음이온 단일 가닥 핵산 사이에 정전기적 반발력을 감소시켜 target에 대한 친화력을 증가시킬 수 있습니다.
ZNA-DNA/ZNA-RNA hybrid의 녹는점을 쉽게 예측하여 Tm값을 oligocation의 길이에 따라 선형적(linear)으로 증가시킵니다.
ZNA를 PCR 및 Reverse Transcription의 primer로 사용하면 specific하고 sensitive한 반응이 가능하며, ZNA probe는 assay design에서 폭넓은 flexibility를 가지며, Minor Groove Binder (MGB)와 Locked Nucleic Acid (LNA)의 효율적인 대체제로 사용될 수 있습니다.

MGB in comparison to ZNA®-conjugated, LNA-modified oligonucleotides

MGB (Minor Groove Binder), ZNA®, LNA(Locked Nucleic Acids), and PNA (Peptide Nucleic Acid) are known to increase the Tm of an oligo sequence.
MGB (Minor Groove Binder) probes include a minor groove binder (MGB) moiety at the 3’ end that increases the melting temperature (Tm) of the probe and stabilizes the hybridization of the probe DNA to its target sequence.
The introduction of the minor groove binder moiety is sequence-independent, meaning the core sequence remains “unmodified”.

ZNA®s are oligonucleotides conjugated with repeated cationic spermine units that decrease electrostatic repulsions with target nucleic acid strands, and greatly improve hybridization properties by enhanced affinity to the complementary target sequence as well as increased stability of the formed duplex at an unprecedented specificity.
The “Tm boost” generated by adding ZNA® to either end of the oligonucleotide probe is significant and also sequence-independent.

In contrast, LNA probes rely on modified nucleotide chemistry with regard to the sugar component involved, while the organic base component is “unmodified” and thus follows Watson-Crick base-pairing rules when mixed with DNA or RNA bases in an oligonucleotide.
When incorporated into an oligonucleotide probe, locked nucleic acid monomers increase structural stability, resulting in a raise of the formed duplex melting temperature (Tm).
Locked Nucleic acids are not recognized by DNA/RNases as a substrate, hence LNA modified oligonucleotides also display significant resistance to nucleases.

In summary, Metabion offers all three duplex stability-enhancing modifications, and therefore provides the greatest flexibility in assay design and choosing the right option for your required application.

• enhanced/accelerated target recognition
• increased sensitivity
• high specificity
• increased Tm for short probes
• improved quenching for long probes
• increased flexibility in terms of
-efficiency at low oligo concentration
-efficiency at high annealing temperatures-
-Mg2+ concentration adjustment
-design/sequence composition constraints
•Probes for post-PCR melting curve analysis
•Enhanced primer/probe sensitivity and specificity through the incorporation of base analog (pdC, pdU)

providing for

• efficient mismatch/SNP/allelic discrimination
• earlier Ct values
• improved quantification accuracy of low-abundance transcripts
• improved RNA to cDNA conversion
• higher signal level
• targeting highly conserved, specific (including A/T rich) sequences
• universal cycling conditions due to ‘Tm leveling’ effects (stabilization of A/T rich duplexes), hence supporting multiplex assays
• An excellent alternative to MGB and LNA

a wide range of Nucleic Acid-based applications like:

• PCR / real-time PCR/RT-PCR
• Microarrays/Capture probing
• Northern Blot / Dot Blot
• In Situ Hybridization (ISH)
• Blocking of alleles (clamp PCR)
• Next-generation sequencing

What we offer

Standard length range between 10 and 40 nucleotides for primers and probes.

For primers, 4 standard ZNA® building block modifications: ZNA-2, ZNA-3, ZNA-4, and ZNA-5!

ZNA-2 for oligos ≥ 8mers
ZNA-3 for oligos ≥ 12mers
ZNA-4 for oligos ≥ 16mers
ZNA-5 for oligos ≥ 20mers

For probes, 3 standard ZNA-Quencher (Q*ZNA-X) building block modifications for 3´labeling (For 5´ ZNA-Reporter labeling, please inquire!);
Respective choice of 3´building blocks will be made automatically by metabion according to the following scheme:

3′ Q*ZNA-2 for probes ≥ 8mers
3′ Q*ZNA-3 for probes ≥ 14mers
3′ Q*ZNA-4 for probes ≥ 18mers

*HPLC and documented Mass-Check QC included
*5´ZNA modified dual labeled probes, introducing 5´reporters like FAM, Fluo, JOE, ROX, TAMRA, etc (see table below)
*All ZNA® oligos will be delivered dissolved in purified distilled water (pH 7-9), with a concentration of 100 µM (for special requests, please inquire)

Delivery (발송)

Metabion normal olgo는 working days 기준 7~10일 정도 소요 되니다.
Modified oligo는 여기에 3-5일  이상 추가 시간이 소요 됩니다.

FAQ

What kind of documentation do I get with my ZNA®?
On the tube label, you will find basic information such as oligo name, name of the person who made the order, oligo sequence including modifications, oligo ID, delivered quantity of DNA, and molecular weight.
In addition, you will receive a technical data sheet including more detailed information on the Physico-chemical properties of the oligo like base composition, GC content, synthesis scale, purification grade, quality control information, etc. You will also receive a printout of the Mass-Check, free of charge.

What concentration of ZNA® primers and probes are recommended for PCR applications?
ZNA® displays high sensitivity in PCR applications, due to enhanced kinetics in target recognition and “scanning”.
This is the reason why ZNA® primers and probes should be used in lower concentrations, compared to other DNA-oligos.
Typical concentrations are 100-200 nM for primers and 200 nM for probes.
A concentration even lower than 50 nM has been proven successful in PCR, without loss of sensitivity.
This may represent a great advantage in multiplex assays, which use multiple sets of primers.

Oligo 보관시 유의점

일반적으로 올리고는 화학적으로 안정하며, -20℃에서 6개월 이상 안정하다고 볼 수 있습니다.
특히, 동결 건조한 상태에서는 매우 안정적이며 오래 보관하실 수 있습니다.
용액 상태에서도 안정하지만, nuclease등의 contamination에 의해 degradation 될 수도 있습니다.

또한, 올리고는 얼렸다 녹였다’를 반복하게 되면 분해를 촉진시켜 품질의 이상이 발생될 수 있습니다.
DNA의 경우는 acidic한 조건에서는 depurination에 의해 decompose될 수 있으니 pH를 낮추지 말아야 합니다.

ZNA primers and probes 주문하는 방법

아래 링크의 올리고 주문양식을 다운로드하여 각 항목을 빠짐없이 작성후 oligo@bio-d.co.kr 로 보내주시면 됩니다.