Ch1. Why Gene Cloning and DNA analysis are important?

- Basic steps in Gene cloning

 

1. A fragment of DNA, containing the gene to be cloned, is inserted into a circular DNA molecule (a vector), to produce a recombinant DNA molecule.

2 The vector transports the gene into a host cell (a bacterium) although other types of living cell can be used.

3 Within the host cell the vector multiplies, producing numerous identical copies, not only of itself but also of the gene that it carries. 

4 When the host cell divides, copies of the recombinant DNA molecule are passed to the progeny and further vector replication takes place.

5 After a large number of cell divisions, a colony, or clone, of identical host cells is produced. Each cell in the clone contains one or more copies of the recombinant DNA molecule. The gene carried by the recombinant molecule is now said to be cloned.

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- Basic steps in the polymerase chain reaction

1. 혼합물의 온도를 94℃까지 올림. 이중 가닥 DNA의 수소결합이 깨짐. 분자가 변성됨. (denature)

2. The mixture is cooled down to 50–60 ◦C. 두 가닥의 분자가 이 온도에서 결합되지만 대부분은 아님. 혼합물은 많은 양의 짧은DNA 분자를 포함하기 때문. called oligonucleotides or primers.

3 The Taq DNA polymerase의 최적 온도인 74◦C까지 올림. Taq DNA polymerase는 각primer의 말단에 결합해 주형 DNA 가닥에 상보적으로 새로운 DNA를 합성해 나감. ∴ 4가닥의 DNA존재.

4. 반복해나감. 겁나 많이 존재.

 

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Why gene cloning and PCR are so important?

 

- Obtaining a pure sample of a gene by cloning

원하는 유전자를 target하기 힘듦. 여러 random한 가닥.

원하는 유전자를 carry하는 vector선별하기 위해 각각을 bacteria에 삽입함.

Batch에서 키워 colony생성.

한 bacteria에서 생긴 colony ➝  유전 정보 동일

 

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- PCR can also be used to purify a gene

: target gene을 잘라내서 vector에 넣는다.

But how can we select or identify just one gene?

 

PCR을 통해 target gene을 증폭시켜 vector 에 삽입.

단점: 최소 양쪽 유전자 서열을 알아야 함.