19.1: DNA sequencing and DNA cloning are valuable tools for genetic engineering and biological inquiry

## *ch 19 The DNA Toolbox*

### 19.1

***19.1: DNA sequencing and DNA cloning are valuable tools for genetic engineering and biological inquiry***

### 19.2

*19.2 Biologists use DNA to study gene expression and function*

### 19.3

*19.3 Cloned organisms and stem cells are useful for basic research and other applications*

### 19.4

*19.4 The practical applications of DNA-based biotechnology affect our lives in many ways*

The complementarity of the two DNA strands is the basis for nucleic acid hybridization (核酸雜合), the base pairing of one strand of nucleic acid to the complementary sequence on another strand

Genetic engineering (基因工程) is the direct manipulation of genes for practical purposes

## DNA Sequencing (DNA定序)

### Frederick Sanger 的 DNA 定序法：Dideoxy termination sequencing

1. DNA 裡 dNMP 與 dNMP 間為磷酯鍵，如果將三號碳的氧也去除(ddNTP)即無法脫水形成鍵結，DNA 複製就會終止。(ddNTP 可作為複製終點)

2. 過程中會產生長短不一的 DNA 片段，加以分離，就能判別出 DNA 的序列了。

### Next-generation sequencing (次世代定序)

如果 dNTP 被加入延長
中的 DNA，則焦磷酸 (PPi) 會釋出，而亮光 則會被記錄

1. 使用一個固定住且被放大過的模板股製造大量相同的 DNA 片段。

2. 數以萬計的 DNA 片段(約 400~1000bps)可以同時被定序。

3. 這是一項高生產量的科技。

## Making Multiple Copies of a Gene or Other DNA Segment

DNA cloning (DNA 轉殖)

The production of multiple copies of a single gene is a type of DNA cloning called **gene cloning (基因選殖)**

## Using Restriction Enzymes to Make Recombinant DNA

* restriction enzyme 限制酶
* restriction sites 限制位置
* restriction fragments (限制片段)
* sticky ends (粘聯端)
* ligase(連接酶)

## gel electrophoresis (膠體電泳)

To check the recombinant plasmid, researchers might cut the products again

using the same restriction enzyme

## Amplifying DNA: The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning

A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

PCR (聚合酶鏈反應), can produce many copies of a specific target segment of DNA

The key to PCR is an unusual, heat-stable DNA polymerase called *Taq* polymerase

PCR 在片段擴大的過程中使用一對(不一定互補)特定的引子。

Another difficulty with eukaryotic gene expression in bacteria is the presence of **introns** in most eukaryotic genes

Researchers can avoid this problem by using **cDNA**, complementary to the mRNA, which contains only exons

## Eukaryotic DNA Cloning and Expression Systems

Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells, such as **yeasts**, as hosts for cloning and expressing genes

cultured mammalian人工培養哺乳類 or insect昆蟲類 cells may be used to express and study proteins

### electroporation (電穿孔技術)

Applying a brief electrical pulse to create temporary holes in plasma membranes introducing recombinant DNA into eukaryotic cells

### microinjection (顯微注射)

Alternatively, scientists can inject DNA into cells using microscopically thin needles

## Cross-Species Gene Expression and Evolutionary Ancestry

The Pax-6 genes in flies and vertebrates can substitute for each other 蒼蠅或脊椎動物的 Pax-6 基因可以互相取代

1. ↑ https://zh.wikipedia.org/wiki/%E5%87%9D%E8%86%A0%E9%9B%BB%E6%B3%B3
2. ↑ https://zh.wikipedia.org/wiki/%E8%81%9A%E5%90%88%E9%85%B6%E9%93%BE%E5%BC%8F%E5%8F%8D%E5%BA%94
3. ↑ https://en.wikipedia.org/wiki/Electroporation
4. ↑ http://zh.biology.wikia.com/wiki/%E9%A1%AF%E5%BE%AE%E6%B3%A8%E5%B0%84%E6%B3%95?variant=zh-tw