Ezway™ Direct PCR Buffer

• PCR전에 DNA를 purification할 필요가 없이 바로 PCR을 수행할 수 있습니다
  • DNA purification에 드는 시간을 절약해 드립니다.
  • 번거로운 스텝을 줄여주므로 편리합니다.
  • 샘플핸들링시 발생할지도 모르는 contamination 위험으로부터 안전합니다.
  • DNA purification중에 발생하는 샘플 loss의 염려가 없습니다.
  • 여러조작이 필요없으므로, 다량의 샘플을 한번에 PCR할 수 있습니다.
• Whole blood뿐 아니라 다양한 샘플에 Direct PCR을 적용할 수 있습니다.
  • 가능한 샘플
    : Whole blood, Dried Blood on papers, Cultured cells, Buccal cells, Saliva, Mouse tail, Paraffinized tissue slide/TMA, Tissue/Organ/Meat, Hair root, Bacteria, Sputum, Bronchial lavage fluid, Urine, Plant, Fungus, Yeast etc.
• 어떤 DNA Polymerase를 이용해도 Direct PCR이 가능합니다.
  • 고마에서 판매되는 Taq외에도 타사의 Taq을 이용하실수 있습니다.
  • Taq, Pfu, Pwo, Tth등의 다양한 DNA polymerase 뿐만 아니라, chemically modified Hot start DNA polymerase와 사용해도 Direct PCR이 가능합니다.
  • AmpliTaq Gold DNA Polymerase (Applied Biosystems사) 이용시에는, 이제품에 specific하게 작용하는 Direct PCR Buffer Gold의 사용을 권장합니다.
• Hot start 효과를 가지므로, background없는 깨끗한 특이 밴드만을 잡아낼 수 있습니다.
• 샘플의 조건에 따라 lysis나 elution이 필요한 경우에 쉽게 사용할수 있도록 Set 타입으로도 제공합니다.

• Forensic science
• Viral infection, Molecular diagnostic test
• Multiplex PCR. SNP detection, PCR-RFLP
• Single cell diagnostic
• Sequencing, Cloning
• Blood banking, Identity testing
• Laboratory automatic PCR

• 제품의 구성
  • Direct PCR Buffer
    • 5X EzWay™ Direct PCR Buffer
  • Direct PCR Buffer Set
    • 5X EzWay™ Direct PCR Buffer
    • 2.5X Direct Lysis Buffer I
    • 1X Direct Lysis Buffer II
    • 4X Magic Buffer (For amplification of high GC contents)

적용예
Fig.1 Direct PCR from whole blood



PCR amplifications were performed using 20 ng human genomic DNA or 1 ul Heparin-treated whole blood in a 50ul PCR reaction volume. EzWayTM Direct PCR Buffer can amplify directly up to 2kb amplicon and achieve clear amplification than DNA PCR. It means that direct PCR amplification has hot start function even without the help of hot start enzyme because the cells are disrupted in initial heating stage, then the exposed DNA templates meet primers instantly at higher temperature than Tm.

Lane1: 146 bp	Lane4: 489 bp	Lane7: 1,191 bp
Lane2: 286 bp	Lane5: 606 bp	Lane8: 1,491
Lane3: 357 bp	Lane6: 817 bp	Lane9: 1,702 bp

Fig.2 Direct PCR from cultured cell

Lane 1 : Control (DNA)

Lane 2 : 3x10

Lane 3 : 3x102

Lane 4 : 3x103

Lane 5 : 3x104v

Lane 6 : 3x105

Lane M : DNA ladder
Direct PCR was performed with U-138 brain tumor cell. Cells were lysed with the EzWayTM Direct Lysis Buffer II first and incubated at room temperature for 10 minutes. 1ul of the supernatant was added directly to 20ul PCR reaction mixtures.
Fig.3 Direct PCR from mouse tail



PCR template was obtained from mouse albino tail by incubation at 56°C for 10 minutes with the EzWayTM Direct Lysis Buffer I and Proteinase K (PK). PK was removed by heating at 95°C for 5 minutes, then,1ul of the supernatant was added directly to 20ul PCR reaction mixtures. The mouse albino mutation was identified by rapid lysis and PCR-RFLP using EzWayTM Direct PCR Buffer. A point mutation in the tyrosinase gene of BALB/c albino mouse at position 85 (cystein → serine) creats an additional DdeI site and this mutation is actually present in BALB/c genomic DNA.
BALB/c strain : 20, 113, 35, 130 bp
C57BL/6 strain : 20, 113, 165 bp

Lane 1, 2, 5, 6, 9, 10 : PCR amplicon

Lane 3, 4, 7, 8, 11, 12 : PCR-RFLP with DdeI

Fig.4 Direct PCR from Plant



Electropherogram with Arabidopsis leaf lysate and purified genomic DNA. Four leaves of Arabidopsis were mixed with 100 ul of 1X Direct Lysis Buffer I, ground with a pestle (+) or not ground (-), followed by incubation at 80°C for 2 hours. After heat treatment, these lysates were amplified with conventional PCR buffer (Lane 1-4) and EzWayTM Direct PCR Buffer (Lane 5-8). Amplification of genomic DNA from two ecotypes,Col (lane 9) and Ler (lane 10), was identified with amplicon sizes of 0.85 and 1.0 kb, respectively. Thus, one sample (lane 1, 3, 5, and 7) was a Col homozygote, and the other (lane 2, 4, 6, and 8) was a Col/Ler heterozygote.

Lane 1-4 : Direct PCR with conventional PCR reaction buffer

Lane 5-8 : Direct PCR with EzWayTM Direct PCR Buffer

Lane M : 100 bp DNA ladder

Fig.5 Melting curve analysis of duplex real-time PCR amplification of S. flexneri and S. typhimurium

The virA and invA gene fragments were amplified simultaneously from fecal samples spiked with equal amounts of S. flexneri and S. typhimurium in the presence of EzWayTM Direct PCR Buffer.
4x105 CFU/ml (●), 4x104 CFU/ml (○), 4x103 CFU/ml (▼),
2x102 CFU/ml (▽), 1x102 CFU/ml (■).
Tms were resolvable between the two products. Tm was 82°C and 87°C for virA and invA, respectively. Specific products are visible at 215 bp and 284 bp for each dilution.

• Direct PCR Technology
• Direct Taq PCR MasterMix
• EzWay™ Direct ApoE Genotyping Kit
• EzWay™ Direct Hot Taq PCR MasterMix
• Mouse Tail Direct PCR Kit
• Plant Direct PCR Kit