Polymerase Chain Reaction Now let's consider how to obtain DNA segments that are suitable for sequencing. At first, DNA sequences were obtained from cloned DNA segments(we will discuss some methods to clone new genes in a subsequent lecture). Presently the entire DNA sequence for E. coli, as well as a variety of other bacterial species, has been determined. If we want to find the sequence of a new mutant allele of a known gene we need an easy way to obtain a quantity of this dna from a culture of bacterial cells. The best way to do this is to use a method known as PCR or polymerase chain reaction that was developed by Kary Mullis in the mid-1980s. The steps in a PCR reaction are as follows (1)A crude preparation of chromosomal DNA is extracted from the bacterial strain of nterest (2) Two short oligo nucleotide primers(each about 18 bases long) are added to the dna The primers are designed from the known genomic sequence to be complimentary to opposite strands of dna and to flank the chromosomal segment of interest (3) The double stranded DNA is melted by heating to 100"C and then the mixture is cooled to allow the primers to anneal to the template dna (4)DNA polymerase and the four nucleotide precursors are added and the reaction is incubated at 37 C for a period of time to allow a copy of the segment to be synthesized (5) Steps 3 and 4 are repeated multiple times. To avoid the inconvenience of having te add new DNa polymerase in each cycle a special DNA polymerase that can withstand heating to 100C is used The idea is that in each cycle of melting, annealing and DNA synthesis the amount of the DNA segment is doubled. This gives an exponential increase in the amount of the specific DNA as the cycles proceed. After 10 cycles the dna is amplified 103 fold and after 20 cycles the dNa will be amplified 10 fold. Usually amplification is continued until all of the nucleotide precursors are incorporated into synthesized dNAPolymerase Chain Reaction Now let’s consider how to obtain DNA segments that are suitable for sequencing. At first, DNA sequences were obtained from cloned DNA segments (we will discuss some methods to clone new genes in a subsequent lecture). Presently the entire DNA sequence for E. coli, as well as a variety of other bacterial species, has been determined. If we want to find the sequence of a new mutant allele of a known gene we need an easy way to obtain a quantity of this DNA from a culture of bacterial cells. The best way to do this is to use a method known as PCR or polymerase chain reaction that was developed by Kary Mullis in the mid-1980’s. The steps in a PCR reaction are as follows. (1) A crude preparation of chromosomal DNA is extracted from the bacterial strain of interest. (2) Two short oligo nucleotide primers (each about 18 bases long) are added to the DNA. The primers are designed from the known genomic sequence to be complimentary to opposite strands of DNA and to flank the chromosomal segment of interest. (3) The double stranded DNA is melted by heating to 100˚C and then the mixture is cooled to allow the primers to anneal to the template DNA. (4) DNA polymerase and the four nucleotide precursors are added and the reaction is incubated at 37˚C for a period of time to allow a copy of the segment to be synthesized. (5) Steps 3 and 4 are repeated multiple times. To avoid the inconvenience of having to add new DNA polymerase in each cycle a special DNA polymerase that can withstand heating to 100˚C is used. The idea is that in each cycle of melting, annealing and DNA synthesis the amount of the DNA segment is doubled. This gives an exponential increase in the amount of the specific DNA as the cycles proceed. After 10 cycles the DNA is amplified 103 fold and after 20 cycles the DNA will be amplified 106 fold. Usually amplification is continued until all of the nucleotide precursors are incorporated into synthesized DNA