FAQ

연구용(Research-grade) 플라스미드의 경우, 기본적으로 다음과 같은 QC 시험을 진행합니다.

• 외관(Appearance): 플라스미드 용액의 성상을 육안으로 검사하여 투명도를 평가하고 입자 유무 및 변색 여부를 확인합니다.
• A260/280: 260 nm(핵산)와 280 nm(단백질)에서의 흡광도를 비교하여 플라스미드 DNA의 순도를 측정합니다. 통상 ~1.8 비율을 고순도 DNA로 판정합니다.
• 아가로스 겔을 이용한 균일성 분석(Homogeneity by Agarose Gel): 아가로스 겔 전기영동을 통해 플라스미드 DNA의 분자량 일관성 및 초나선(Supercoiled) 비율을 분석하여 균일성을 평가합니다.
• 제한효소 절단 분석(Restriction Analysis): 특정 제한효소로 플라스미드 DNA를 절단한 후 겔 전기영동으로 단편을 분석하여 플라스미드의 동일성(Identity) 및 온전성(Integrity)을 검증합니다.
• LAL 기반 엔도톡신 시험(Endotoxin by LAL): Limulus Amebocyte Lysate(LAL) 시험법을 통해 플라스미드 조제물 내 세균성 엔도톡신을 검출하여 민감한 실험 및 응용 분야에 안전한지 확인합니다.

또한 고객의 요청에 따라 추가 QC 시험을 제공하고 있으며, 해당 항목들은 당사의 전임상(Preclinical) 플라스미드 품질 관리에도 기본 포함됩니다.

• HPLC를 이용한 균일성 분석(Homogeneity by HPLC): 고성능 액체 크로마토그래피(HPLC)를 통해 플라스미드 DNA의 균일성과 순도를 평가하고, 초나선(Supercoiled) 플라스미드 DNA의 비율을 정밀하게 측정합니다.
• SYBR Gold 염색을 이용한 잔류 RNA 분석(Residual RNA by SYBRGold): SYBR Gold로 염색한 후 형광 강도를 분석하여 플라스미드 조제물 내 잔류 RNA를 정량합니다.
• qPCR을 이용한 잔류 대장균(E. coli) DNA 분석(Residual E. coli DNA by qPCR): 정량적 PCR(qPCR)을 통해 플라스미드 조제물 내 잔류 대장균 유전체 DNA(gDNA)를 검출 및 정량합니다.
• 직접 접종법 기반 생균수 시험(Bioburden Testing by Direct Inoculation): 시료를 배지에 직접 접종한 후 미생물 증식 여부를 모니터링하여 미생물 오염도를 평가합니다.
• 생거 시퀀싱(Sequencing by Sanger): 생거 염기서열 분석법(Sanger sequencing)을 통해 삽입 서열(Insert) 또는 플라스미드 전체 염기서열의 정확성을 확인합니다.
• ELISA 기반 잔류 숙주 단백질 분석(Residual Host Protein by ELISA): 대장균 단백질에 특이적인 ELISA 시험법을 사용하여 플라스미드 조제물 내 남아 있는 숙주 유래 단백질(HCP)을 검출합니다.
• qPCR을 이용한 마이코플라스마 오염 검사(Mycoplasma Contamination by qPCR): 고감도 qPCR 기법을 적용하여 플라스미드 시료의 마이코플라스마 오염 여부를 스크리닝합니다.
• 전위차법을 이용한 pH 측정(pH by Potentiometry): 플라스미드 용액의 pH를 측정하여 안정성 및 적용에 적합한 허용 기준 내에 있는지 확인합니다.
• ELISA 기반 잔류 카나마이신 분석(Residual Kanamycin by ELISA): 플라스미드 선별 과정에서 사용된 카나마이신(Kanamycin) 항생제의 잔류 여부를 ELISA 시험법으로 검출합니다.
• 무균 시험(Sterility): 플라스미드 조제물 내 생존 가능한 미생물이 존재하지 않음을 확인하여 무균 상태를 입증하고 민감한 응용에 적합한지 검증합니다.
• 삼투압 측정(Osmolality): 플라스미드 용액의 삼투압 농도(용질 농도)를 측정하여 생물학적 적합성 허용 범위 내에 있는지 확인합니다.

We use seamless cloning to construct most plasmids.

Seamless cloning is a method that allows precise insertion of DNA fragments into plasmid vectors without adding extra nucleotides at the junctions, a common issue with traditional restriction enzyme methods. This is crucial for protein expression, as even small additional amino acids can affect protein function. There are various approaches to seamless cloning, including techniques like overlap extension PCR or commercial kits.

Key Steps in Seamless Cloning:
Creation of Overlapping Ends: The DNA fragments are generated through PCR with ends that overlap with each other or the plasmid. These overlaps are designed into the primers used for amplification.
Annealing of Overlapping Ends: The complementary overlapping regions hybridize when mixed together.

Extension and Ligation:
A polymerase may extend the annealed fragments, filling in gaps.
DNA ligase then seals the nicks, or a commercial system may combine both enzymatic steps in a single process.

Requirements:
DNA Fragments with Overlaps: These fragments, usually created by PCR, must have complementary sequences for hybridization.
Polymerase: A polymerase is needed for filling in gaps, if required.
DNA Ligase: Ligase seals the nicks unless using a system that combines all steps.

Pros and Cons:

Pros:
Enables precise, in-frame gene insertions without unwanted nucleotides.
No need for restriction enzyme sites, giving more design flexibility.
Simplified primer design.

Cons:
Primer design requires precision for correct overlap and orientation.
Efficiency can depend on factors like fragment size and sequence complexity.
Commercial kits can be expensive.

Tips and Tricks:
Optimal Overlap Length: Overlaps of 15–25 nucleotides typically provide efficient annealing, though larger fragments may require longer overlaps.
High Purity DNA: DNA fragments should be pure, often achieved by gel purification post-PCR, for optimal results.
Control Reactions: Always include controls (like no-insert controls) to detect potential background noise or unwanted ligation.

We use the strains below depend on different applications:

1. DH5α
Genotype: F–, φ80dlacZΔM15, Δ(lacZYA-argF)U169, recA1, endA1, hsdR17(rK–, mK+), phoA, supE44, λ–, thi-1, gyrA96, relA1
Applications: Commonly used for general cloning purposes and blue/white screening. Its mutations in recA and endA enhance plasmid stability and transformation efficiency, making it a go-to strain for many cloning applications.

2. Top10
Genotype: F–, mcrA, Δ(mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, recA1, araD139, Δ(ara-leu)7697, galU, galK, λ–, rpsL(StrR), endA1, nupG
Applications: Suitable for general cloning and blue/white screening with high transformation efficiency. It is often preferred when maximizing the number of transformants is critical.

3. Stbl3
Genotype: F–, mcrB, mrr, hsdS20(rB–, mB–), recA13, supE44, ara-14, galK2, lacY1, proA2, rpsL20(StrR), xyl-5, λ–, leu, mtl-1
Applications: Ideal for cloning vectors with repetitive elements, such as long terminal repeats (LTRs) in lentiviral plasmids. The recA mutation minimizes recombination, enhancing the stability of complex constructs.

4. XL-10
Genotype: TetR Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte [F´ proAB lacIqZΔM15 Tn10 (TetR) Amy CamR]
Applications: Optimized for high-efficiency transformation, particularly for large or methylated DNA constructs. It is also suitable for blue/white screening and for cloning unstable or toxic sequences due to the recA mutation.

5. NEB Stable
Genotype: F´ proA+B+ lacIq Δ(lacZ)M15 Tn10 (TetR) endA1 recA1 hsdR17(rK– mK+) glnV44 λ– thi-1 gyrA96 relA1 spoT1
Applications: Designed to maintain unstable plasmids that might recombine or degrade in other strains. It is ideal for cloning large or recombination-prone plasmids, offering good yield and stability for long-term propagation.

An Antibiotic Resistance Gene in a plasmid provides the host cell with the ability to survive in the presence of a specific antibiotic. This is used as a selection marker to ensure that only cells that have successfully taken up the plasmid (transformed cells) will grow in an antibiotic-containing environment. Common antibiotic resistance genes include AmpR (confers resistance to ampicillin) and KanR (confers resistance to kanamycin). Researchers use antibiotic selection to isolate and maintain populations of cells containing the plasmid.

폴리아데닐화 시그널(Polyadenylation Signal, Poly(A) 시그널)은 유전자의 말단 부근에 위치하는 염기서열로, 전사(Transcription) 과정 중 mRNA의 끝부분에 폴리(A) 꼬리(Poly(A) tail)가 부가되도록 지시하는 역할을 합니다.

이 폴리(A) 꼬리는 mRNA의 안정성을 유지하고 단백질로의 번역 효율을 높이는 데 핵심적인 역할을 담당합니다. 또한 mRNA가 핵(Nucleus)에서 세포질(Cytoplasm)로 원활하게 수송(Export)되도록 돕습니다. 따라서 플라스미드(Plasmid) 벡터에 Poly(A) 시그널을 포함하면 목적 유전자(Gene of interest)로부터 전사된 mRNA가 정상적으로 가공(Processing)되어, 한층 더 안정적이고 효율적으로 단백질을 발현시킬 수 있습니다.

Regulatory elements are sequences within the plasmid that help control the expression of the gene of interest. One common regulatory element is the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), which enhances the stability and translation efficiency of mRNA. WPRE is often included in viral vectors or plasmids to increase gene expression, leading to higher levels of protein production in the host cells. Other regulatory elements may control transcription rates, splicing, or mRNA transport.

An Open Reading Frame (ORF) is a continuous sequence of codons in DNA that starts with a start codon (usually AUG) and ends with a stop codon (such as UAA, UAG, or UGA). The ORF represents the portion of a gene that is translated into a protein. In plasmid design, the ORF usually includes the gene of interest that researchers wish to express in a host organism. Proper expression of the ORF is crucial for the successful production of the encoded protein.

The Multiple Cloning Site (MCS), also known as a polylinker, is a short segment of DNA that contains several unique restriction enzyme recognition sites. These sites allow researchers to insert a gene of interest into the plasmid at a specific location. The MCS simplifies the process of cloning because it offers flexibility in choosing which restriction enzymes to use, making it easier to incorporate foreign DNA fragments into the plasmid without disrupting other essential elements.

Today, seamless cloning methods are often preferred over using restriction enzymes in the MCS for gene insertion. However, the restriction enzyme sites within the MCS still provide a convenient location for plasmid linearization during seamless cloning.