Polymerase Chain Reaction (PCR)

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PCR是一项获得了诺贝尔奖的重要技术主要用于针对性的扩增DNA片段
一 PCR的过程

Denaturation at 94℃（变性）

DNA双链模板在该温度会变为单链
模板是怎么获得的？

Annealing at 54℃（退火）
引物和DNA单链结合聚合酶就可以找到要延伸的位置
为什么需要引物？

Extension at 72℃（延伸）
聚合酶的协助下将核苷酸dATP, dCTP, dGTP,dTTP 从5‘->3‘ 方向使用互补原则开始延伸

三个关键元素
Primers are short pieces of DNA that are made in a laboratory. Since they're custom built, primers can have any sequence of nucleotides you'd like.

In a PCR experiment, two primers are designed to match to the segment of DNA you want to copy. Through complementary base pairing, one primer attaches to the top strand at one end of your segment of interest, and the other primer attaches to the bottom strand at the other end. In most cases, 2 primers that are 20 or so nucleotides long will target just one place in the entire genome.

Primers are also necessary because DNA polymerase can't attach at just any old place and start copying away. It can only add onto an existing piece of DNA.

DNA Polymerase is a naturally occurring complex of proteins whose function is to copy a cell's DNA before it divides in two. When a DNA polymerase molecule bumps into a primer that's base-paired with a longer piece of DNA, it attaches itself near the end of the primer and starts adding nucleotides. (In nature, these primers are made by an enzyme called primase).

The DNA polymerase in our bodies breaks down at temperatures well below 95 °C (203 °F), the temperature necessary to separate two complementary strands of DNA in a test tube. The DNA polymerase that's most often used in PCR comes from a strain of bacteria calledThermus aquaticus that live in the hot springs of Yellowstone National Park. It can survive near boiling temperatures and works quite well at 72 °C (162 °F).

Nucleotides are the building blocks that DNA molecules are made of. You add a mixture of four types of nucleotides to your PCR reactionA's, C's, G's and T's. DNA polymerase grabs nucleotides that are floating in the liquid around it and attaches them to the end of a primer.
二 PCR的限制
2.1 聚合酶错误
当前主流的是Taq polymerase，该酶无法保证延伸时的碱基是否正确
2.2 长度受限
通常只能扩增 2-3Kb
2.3 非特异性引物（Non-specific Priming）
引物设计不合理导致扩增的片段不是希望扩增的片段

三改进
RT-PCR

四应用场景

参考资料：
1.https://thebiologynotes.com/polymerase-chain-reaction-pcr/
2.https://en.wikipedia.org/wiki/Polymerase_chain_reaction
3.https://learn.genetics.utah.edu/content/labs/pcr/
https://learn.genetics.utah.edu/content/labs/交互式实验
4.https://www.britannica.com/science/polymerase-chain-reaction