Appendix A: 

 Primer on Molecular 

 Genetics 



In vitro cloning. A new approach to cloning, the polymerase chain reaction (PCR) 

 amplifies DNA molecules in the test tube without using a host cell (Fig. 8). PCR is 

 especially valuable because the reaction is easily automated and can handle samples too 

 small or too to.xic to be managed inside cells. 



If a set of DNA fragments contains a complete sample of an entire genome or 

 chromosome, it is called a library (Figs. 7 and 9). 



Fig. 8. DNA amplification by the 

 polymerase chain reaction (PCR). 



PCR is a recently developed 

 method for cloning human DNA — a 

 necessary prelude to any intensive 

 mapping or sequencing efforl. PCR 

 begins with the annealing of primers 

 to the single-stranded fragments 

 that are to be replicated. With the 

 primers attached, a DNA replication 

 enzyme can complete the 

 complementary strands thus 

 started. The succeeding PCR cycle 

 then operates on the newly 

 produced duplicates, as well as the 

 original fragments. Each cycle thus 

 doubles the amount of the selected 

 DNA fragments in the reaction 

 mixture. Twenty-five cycles, which 

 can be completed in less than three 

 hours, can theoretically produce 

 amplification by 30 million-fold: in 

 practice, amplification by a factor of 

 over one hundred thousand can be 

 readily achieved. 



,i 7 \t'<i>,:l, ilr<imli. 



\r^7 Extend complementary strands 

 ^-^ by emymatK rephcatton 



^^4/ ffepedl steps I and 2 



128 



