📚 A-Level Biology: Key Tools and Enzymes in Gene Technology | A-Level 生物：基因技术常用工具与酶
Gene technology, also known as genetic engineering, involves the manipulation of DNA to alter the characteristics of an organism. This process relies on a suite of molecular tools, including restriction enzymes, ligases, polymerases, and various vectors, each performing a specific function in the construction and delivery of recombinant DNA molecules.
基因技术又称基因工程，是指对 DNA 进行操作以改变生物性状的科学。该过程依赖一系列分子工具，包括限制性内切酶、连接酶、聚合酶以及各类载体，它们在重组 DNA 分子的构建与递送中各司其职。
1. Restriction Enzymes | 限制性内切酶
Restriction enzymes (or restriction endonucleases) are bacterial enzymes that recognise short, specific, usually palindromic DNA sequences and cut the phosphodiester backbone at or near these sites. For example, EcoRI recognises 5′-GAATTC-3′ and cuts between the G and A on both strands, generating sticky ends with overhanging single-stranded regions.
限制性内切酶（或限制性核酸内切酶）是细菌产生的一类酶，能够识别通常成回文结构的特异性短 DNA 序列，并在其内部或附近切割磷酸二酯键骨架。例如，EcoRI 识别 5′-GAATTC-3’，在两条链的 G 与 A 之间切割，产生带有单链突出段的黏性末端。
Sticky ends are valuable in gene cloning because they can hydrogen-bond with complementary overhangs on other DNA fragments. Blunt-end cuts produce no overhangs and are harder to ligate efficiently. The cell’s own DNA is methylated to protect against its restriction enzymes.
黏性末端在基因克隆中非常重要，因为它们能与另一 DNA 片段的互补突出端形成氢键。平末端切割不产生突出端，因此连接效率较低。细菌自身的 DNA 会被甲基化修饰，从而免受本身限制酶的攻击。
2. DNA Ligase | DNA连接酶
DNA ligase is an essential enzyme that joins DNA fragments by catalysing the formation of phosphodiester bonds between the 3′-hydroxyl group of one nucleotide and the 5′-phosphate group of another. In genetic engineering, it is used to insert a DNA fragment into a vector by sealing the sugar-phosphate backbone after the fragment has annealed to complementary sticky ends.
DNA 连接酶是一种关键酶，它通过催化一个核苷酸的 3′-羟基与另一个核苷酸的 5′-磷酸基团之间形成磷酸二酯键，从而将 DNA 片段连接起来。在基因工程中，当 DNA 片段与互补的黏性末端退火后，连接酶负责封闭糖-磷酸骨架，将片段插入载体中。
This enzyme requires ATP (or NAD+ in some bacteria) as an energy source. For successful ligation, the ends being joined must be correctly aligned; sticky ends enhance both specificity and efficiency compared with blunt ends, which require higher DNA concentrations and more enzyme.
该酶需要 ATP（某些细菌中也可用 NAD⁺）作为能量来源。要成功连接，待拼接的末端必须正确对齐。与平末端相比，黏性末端既能提高特异性也提升效率，而平末端连接需要更高的 DNA 浓度和更多的酶。
3. Reverse Transcriptase | 逆转录酶
Reverse transcriptase is an enzyme that synthesises a single-stranded DNA molecule from an RNA template. In gene technology, it is used to make complementary DNA (cDNA) from eukaryotic messenger RNA (mRNA). Because the mRNA has already undergone splicing, the resulting cDNA lacks introns and can be expressed in bacterial hosts.
逆转录酶是一种以 RNA 为模板合成单链 DNA 分子的酶。在基因技术中，它常被用于从真核生物的信使 RNA（mRNA）制备互补 DNA（cDNA）。由于 mRNA 已经过剪接，所得 cDNA 不含有内含子，因而可在细菌宿主中表达。
The typical reaction involves an oligo(dT) primer that anneals to the poly-A tail of mRNA, allowing reverse transcriptase to extend the primer and produce an RNA-DNA hybrid. The RNA strand is then removed and a second DNA strand is synthesised by DNA polymerase, yielding double-stranded cDNA for cloning.
典型反应利用一段 oligo(dT) 引物与 mRNA 的 poly-A 尾互补退火，逆转录酶随后延伸引物生成 RNA-DNA 杂交双链。接着 RNA 链被去除，DNA 聚合酶再合成第二条 DNA 链，得到可用于克隆的双链 cDNA。
4. DNA Polymerase and Taq Polymerase | DNA聚合酶与Taq聚合酶
DNA polymerase is the enzyme that synthesises new DNA strands by adding nucleotides complementary to a template strand. In genetic engineering, DNA polymerase is used in techniques such as PCR and in filling in overhangs to create blunt ends. Standard DNA polymerases from E. coli have 5’→3′ polymerase activity and often also 3’→5′ proofreading activity.
DNA 聚合酶是一种通过读取模板链合成新 DNA 链的酶。在基因工程中，它用于 PCR 等技术，也可用于填补突出端以形成平末端。来自大肠杆菌的标准 DNA 聚合酶具有 5’→3′ 聚合酶活性，通常还兼具 3’→5′ 校对活性。
Taq polymerase, isolated from Thermus aquaticus, is thermostable and can withstand the 95 °C denaturation step of PCR. However, it lacks 3’→5′ proofreading activity, so it introduces errors more readily than proofreading polymerases such as Pfu. The ability to work at 72 °C extension makes Taq polymerase the standard choice for routine PCR.
Taq 聚合酶分离自水生栖热菌，具有热稳定性，能耐受 PCR 中 95 °C 的变性步骤。但它缺乏 3’→5′ 校对活性，因此相比 Pfu 等校对型聚合酶更容易引入错误。由于它能在 72 °C 下进行延伸，Taq 聚合酶成为常规 PCR 的首选。
5. Plasmids as Vectors | 质粒载体
A plasmid is a small, circular, double-stranded DNA molecule that replicates independently of the chromosomal DNA. In gene technology, plasmids are commonly used as vectors to carry foreign genes into host cells, usually bacteria. An ideal plasmid vector contains several key features: an origin of replication (ori) for autonomous replication, a multiple cloning site (MCS) with unique restriction sites, and selectable marker genes such as antibiotic resistance genes.
质粒是一种独立于染色体 DNA 复制的小型环状双链 DNA 分子。在基因技术中，质粒常被用作载体，将外源基因导入宿主细胞（通常为细菌）。理想的质粒载体具有几个关键特征：用于自主复制的复制起点（ori）、含多个独特限制酶位点的多克隆位点（MCS），以及诸如抗生素抗性基因等选择标记基因。
The MCS allows a foreign DNA fragment to be inserted using the same restriction enzyme used to cut both vector and target DNA. The selectable marker helps identify cells that have taken up the recombinant plasmid; for example, cells containing the plasmid can survive on medium with ampicillin, whereas cells without it cannot.
多克隆位点允许使用同一种限制酶对载体和目标 DNA 进行切割，从而插入外源片段。选择标记有助于识别已摄入重组质粒的细胞；例如，含质粒的细胞能在含氨苄青霉素的培养基上存活，而无质粒的细胞则不能。
6. Other Cloning Vectors | 其他克隆载体
Although plasmids are versatile, other vectors are used for cloning larger DNA fragments or for different host cells. Bacteriophage λ vectors can carry up to 20 kb of foreign DNA; cosmids, which combine plasmid and phage features, accommodate up to 45 kb. For very large fragments, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) can clone >100 kb and >1000 kb respectively.
尽管质粒应用广泛，但其他载体可用于克隆更大的 DNA 片段或用于不同的宿主细胞。噬菌体 λ 载体可携带约 20 kb 的外源 DNA；黏粒（cosmid）结合了质粒和噬菌体的特性，可容纳约 45 kb 片段。对于超大片段，细菌人工染色体（BAC）和酵母人工染色体（YAC）分别可克隆超过 100 kb 和 1000 kb 的 DNA。
| Vector | Insert capacity | Typical host |
| Plasmid | Up to 10 kb | Bacteria |
| Bacteriophage λ | Up to 20 kb | Bacteria |
| Cosmid | Up to 45 kb | Bacteria |
| BAC | 100-300 kb | Bacteria |
| YAC | >1000 kb | Yeast |
Choosing the correct vector depends on the target fragment size and the downstream application. For example, BACs are widely used in genome sequencing projects because they can maintain very long contiguous DNA fragments with low rearrangement rates.
选择何种载体取决于目标片段大小以及下游应用。例如，BAC 因能稳定保持很长的连续 DNA 片段且重组率低，被广泛用于基因组测序项目。
7. PCR (Polymerase Chain Reaction) | 聚合酶链式反应
The polymerase chain reaction (PCR) is a technique used to amplify a specific DNA sequence millions of times. The reaction mixture contains the DNA template, two primers, free nucleotides (dNTPs), Taq polymerase, and Mg²⁺ ions as cofactors. Each cycle consists of three stages: denaturation at 95 °C to separate double-stranded DNA, annealing at around 55 °C to allow primers to bind, and extension at 72 °C for Taq polymerase to synthesise complementary strands.
聚合酶链式反应（PCR）是一种将特定 DNA 序列扩增数百万倍的技术。反应体系包含 DNA 模板、一对引物、游离脱氧核苷酸（dNTPs）、Taq 聚合酶以及作为辅助因子的 Mg²⁺ 离子。每个循环包括三个步骤：95 °C 变性使双链 DNA 解开，约 55 °C 退火使引物结合，72 °C 延伸使 Taq 聚合酶合成互补链。
Primers are short, single-stranded DNA sequences (typically 18-25 nucleotides long) that are complementary to the regions flanking the target sequence. Because PCR amplifies exponentially, after about 30 cycles the target DNA is amplified by a factor of roughly 2³⁰, which is sufficient for further analysis such as gel electrophoresis or DNA sequencing.
引物是短的单链 DNA 序列（通常为 18-25 个核苷酸），与目标区域两侧的序列互补。由于 PCR 呈指数扩增，经过约 30 个循环后，目标 DNA 可扩增约 2³⁰ 倍，足以用于凝胶电泳或 DNA 测序等后续分析。
8. Gel Electrophoresis | 凝胶电泳
Gel electrophoresis is a tool used to separate DNA fragments according to their size. DNA is loaded into wells of an agarose gel and an electric current is applied. Because the phosphate backbone of DNA carries a negative charge, DNA fragments migrate toward the positive electrode. Smaller fragments move through the gel matrix faster than larger ones, resulting in a series of bands.
凝胶电泳是一种按大小分离 DNA 片段的技术。将 DNA 加入琼脂糖凝胶的样品孔中，施加电场。由于 DNA 的磷酸骨架带负电，片段会向正极移动。小片段比大片段在凝胶基质中移动更快，从而形成一系列条带。
The gel is stained with a fluorescent dye such as ethidium bromide or a safer alternative, allowing DNA bands to be visualised under UV light. A DNA ladder (known-size markers) is run alongside samples to estimate fragment sizes. In genetic engineering, gel electrophoresis is commonly used to check whether a restriction digest produced the expected fragments.
凝胶用溴化乙锭或更安全的荧光染料染色后，可在紫外光下观察 DNA 条带。将已知大小的 DNA 标准品（DNA ladder）与样品一同电泳，可估算片段大小。在基因工程中，凝胶电泳常用于检查限制酶酶切是否产生了预期片段。
9. DNA Probes and Hybridisation | DNA探针与杂交
A DNA probe is a single-stranded DNA or RNA fragment that is complementary to a specific target sequence and is labelled with a radioactive or fluorescent marker. In gene technology, probes are used to detect the presence of a particular gene or DNA sequence in a sample. The target DNA is first separated by gel electrophoresis, transferred to a membrane (Southern blotting), and then exposed to the probe under conditions that favour hydrogen bonding between complementary sequences.
DNA 探针是带有放射性或荧光标记的单链 DNA 或 RNA 片段，与特定靶序列互补。在基因技术中，探针可用于检测样品中是否存在特定基因或 DNA 序列。首先通过凝胶电泳分离目标 DNA，将其转移到膜上（Southen 印迹），再在有利于互补序列形成氢键的条件下与探针杂交。
Hybridisation confirms complementarity, and the labelled probe signals the location of the target sequence. This technique is also applied in DNA microarrays, where thousands of probes are fixed to a solid surface to analyse gene expression or detect genetic mutations.
杂交结果证实了互补性，标记探针指示出靶序列的位置。该技术也应用于 DNA 微阵列，即将成千上万种探针固定在固相表面，用于分析基因表达或检测基因突变。
10. CRISPR-Cas9 Gene Editing | CRISPR-Cas9基因编辑
CRISPR-Cas9 is a recent and powerful gene-editing tool adapted from a natural bacterial defence system against viruses. The system uses a guide RNA (sgRNA) that is complementary to a target DNA sequence. The sgRNA binds to the Cas9 nuclease, guiding it to the exact genomic location where Cas9 makes a double-strand break in the DNA.
CRISPR-Cas9 是一种近年发展的强大基因编辑工具，源自细菌抵御病毒的天然防御系统。该系统利用一段与靶 DNA 序列互补的导向 RNA（sgRNA）。sgRNA 与 Cas9 核酸酶结合，引导其到达基因组中的精确位置，由 Cas9 在 DNA 上制造双链断裂。
After the cut, the cell’s natural DNA repair mechanisms are exploited. In non-homologous end joining (NHEJ), the break is repaired with insertions or deletions that can disrupt a gene. In homology-directed repair (HDR), a donor DNA template can be provided to introduce a specific mutation or replace a gene. CRISPR-Cas9 has revolutionised genetics, enabling rapid and precise modification of genes in many organisms.
断裂后，细胞的天然 DNA 修复机制被加以利用。在非同源末端连接（NHEJ）中，断裂以插入或缺失的方式修复，可能破坏基因功能。在同源定向修复（HDR）中，可提供供体 DNA 模板来引入特定突变或替换基因。CRISPR-Cas9 彻底改变了遗传学，使得在多种生物中快速、精确地修饰基因成为可能。
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