Molecular Biology — Biotech & Common Comparisons

1.PCR, polymerase chain reaction, amplifies a specific DNA fragment from tiny starting material. Three step cycle: One, denaturation at ninety-four to ninety-eight degrees — separates double-stranded DNA. Two, annealing at fifty to sixty-five degrees — primers bind the template. Three, extension at seventy-two degrees — Taq DNA polymerase synthesizes new strands.

PCR 聚合酶連鎖反應從微量 DNA 擴增特定片段。三步驟循環：一、變性 94 到 98 度——打開雙股 DNA。二、退火 50 到 65 度——引子結合模板。三、延伸 72 度——Taq DNA 聚合酶合成新股。

2.After n cycles, DNA quantity equals starting amount times two to the power of n. Taq polymerase comes from the thermophilic bacterium Thermus aquaticus — heat-resistant, survives ninety-four degree denaturation across cycles.

經 n 次循環後，DNA 量等於起始量乘以 2 的 n 次方。Taq 聚合酶來自嗜熱菌 Thermus aquaticus，耐高溫，在 94 度變性下能存活多次循環。

3.Applications: COVID-19 testing uses RT-PCR — reverse transcribes RNA to cDNA first, then amplifies. Forensics amplifies trace crime scene DNA. Medicine uses it for prenatal diagnosis of genetic diseases.

應用：COVID-19 檢測用 RT-PCR——先反轉錄 RNA 為 cDNA 再擴增。法醫擴增微量犯罪現場 DNA。醫學用於產前遺傳疾病診斷。

4.Restriction enzymes, also called restriction endonucleases, come from bacterial defense systems. They recognize specific palindromic sequences and cleave them. Sticky ends have staggered cuts with single-stranded overhangs — easy to recombine via complementary pairing. Blunt ends cut evenly — harder to ligate but more versatile.

限制酶又稱限制內切酶，來自細菌防禦系統。辨認特定回文序列並切割。黏性末端切割錯開、留下單股突出——便於互補配對重組。平滑末端切割齊平——較不易接合但更通用。

5.Gel electrophoresis: DNA carries negative charge from phosphates, migrates toward the positive electrode in an electric field. Smaller fragments migrate faster. Agarose gel for DNA; polyacrylamide PAGE for proteins.

凝膠電泳：DNA 由磷酸基團帶負電，在電場中往正極移動。較小片段移動較快。洋菜膠用於 DNA；聚丙烯醯胺膠 PAGE 用於蛋白質。

6.Gene cloning steps: One, cut target gene and vector with restriction enzymes. Two, insert gene into vector using DNA ligase. Three, transform recombinant vector into host bacteria. Four, screen for bacteria containing the target gene, often using antibiotic resistance.

基因選殖步驟：一、用限制酶切割目標基因和載體。二、用 DNA 連接酶將基因插入載體。三、將重組載體轉形入宿主細菌。四、篩選含目標基因的細菌，常用抗生素抗性篩選。

7.CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. It originated as a bacterial adaptive immune system against viruses. Bacteria store fragments of past viral DNA between palindromic repeats and use them to recognize and cut returning invaders.

CRISPR 全名為叢集規律間隔短回文重複序列。源自細菌對抗病毒的適應性免疫系統。細菌將過去病毒 DNA 片段儲存在回文重複序列之間，用以辨識並切割再次入侵的病毒。

8.Cas9 is the molecular scissors protein. Guide RNA, abbreviated gRNA, directs Cas9 to a specific target DNA sequence by complementary base pairing. After binding, Cas9 cleaves the double-stranded DNA at that exact location.

Cas9 是分子剪刀蛋白。導引 RNA，簡稱 gRNA，以互補配對引導 Cas9 到特定目標 DNA 序列。結合後，Cas9 在該精確位置切割雙股 DNA。

9.Two repair pathways after cutting. NHEJ, non-homologous end joining, produces insertion or deletion mutations — used for gene knockout to study gene function. HDR, homology-directed repair, allows precise insertion or replacement of a sequence — used for gene therapy and correcting mutations.

切割後有兩種修復路徑。NHEJ 非同源末端連接，產生插入或缺失突變——用於基因剔除研究基因功能。HDR 同源導向修復，允許精確插入或替換序列——用於基因治療和修正突變。

10.Clinical use: in twenty-twenty, CRISPR inventors Jennifer Doudna and Emmanuelle Charpentier received the Nobel Prize in Chemistry. CRISPR has entered clinical trials for treating sickle cell anemia and beta-thalassemia.

臨床應用：2020 年 CRISPR 發明者 Jennifer Doudna 和 Emmanuelle Charpentier 獲得諾貝爾化學獎。CRISPR 已進入臨床試驗，用於治療鐮刀型貧血和 β 地中海貧血。

11.RNA interference, or RNAi, is a separate gene-silencing technology often paired with CRISPR in essay questions. Small interfering RNAs, siRNAs, bind to complementary mRNA and trigger degradation by the RISC complex — silencing the gene without altering DNA. CRISPR edits the gene; RNAi knocks down its expression.

RNA 干擾即 RNAi 是另一個基因靜默技術，申論常與 CRISPR 一起考。小干擾 RNA 即 siRNA 與互補 mRNA 結合，觸發 RISC 複合體降解——靜默基因但不改變 DNA。CRISPR 編輯基因，RNAi 抑制基因表現。

12.DNA uses deoxyribose sugar; RNA uses ribose. DNA bases A T G C; RNA bases A U G C — uracil replaces thymine. DNA is usually double-stranded; RNA usually single-stranded. DNA is mostly in the nucleus; RNA is in nucleus and cytoplasm. DNA is more stable because deoxyribose lacks the two-prime hydroxyl.

DNA 用去氧核糖；RNA 用核糖。DNA 鹼基是 A T G C；RNA 是 A U G C——U 取代 T。DNA 通常雙股；RNA 通常單股。DNA 主要在細胞核；RNA 在細胞核與細胞質。DNA 較穩定，因為去氧核糖少了 2-prime 氫氧基。

13.DNA replication uses both strands as templates and produces DNA via DNA polymerase, requiring an RNA primer, with three-prime to five-prime proofreading. Transcription uses only the template strand and produces RNA via RNA polymerase, needs no primer, has no proofreading.

DNA 複製兩股都當模板，由 DNA 聚合酶產生 DNA，需要 RNA 引子，有 3-prime 到 5-prime 校對。轉錄只用模板股，由 RNA 聚合酶產生 RNA，不需要引子，無校對。

14.Prokaryotes have one type of RNA polymerase; eukaryotes have three types — Pol one, two, three. Prokaryotic mRNA is not processed and is translated immediately — transcription and translation are coupled. Eukaryotic mRNA undergoes five-prime cap, poly-A tail, and splicing — transcription happens in nucleus, translation in cytoplasm. Prokaryote mRNA is polycistronic; eukaryote mRNA is monocistronic. Prokaryote initiator amino acid is fMet; eukaryote is Met.

原核有 1 種 RNA 聚合酶；真核有 3 種 Pol I、II、III。原核 mRNA 不加工直接翻譯——轉錄轉譯偶聯。真核 mRNA 有 5-prime cap、poly-A 尾、剪接——轉錄在核內，翻譯在核外。原核 mRNA 多順反子；真核單順反子。原核起始胺基酸 fMet；真核 Met。

15.lac operon handles lactose catabolism — inducible, default OFF, induced by allolactose, with cAMP-CAP positive regulation. trp operon handles tryptophan synthesis (anabolism) — repressible, default ON, corepressed by tryptophan, with attenuation as additional regulation.

lac operon 負責乳糖分解——可誘導，預設 OFF，由異乳糖誘導，有 cAMP-CAP 正調控。trp operon 負責色胺酸合成——可抑制，預設 ON，由色胺酸輔抑制，額外調控為弱化機制。