working knowledge

TEMPLATE STRAND dNTP POLYMERASE a b c e d PRIMER POLYMERASE Working Knowledge 112 Scientific American May 1998 WORKING KNOWLEDGE POLYMERASE CHAIN REACTION by Elizabeth A. Dragon Roche Molecular Systems P olymerase chain reaction (PCR) is a technique that mimics nature’s way of replicating DNA. First described in 1985, PCR has been adopted as an essential research tool because it can take a minute sample of genetic material and duplicate enough of it for study. PCR has been used to identify the remains of Desert Storm casualties, to analyze prehistoric DNA, to diagnose diseases and to help make identifications in police investigations. DNA is most often found as a double-stranded molecule, twisted as a helix, in which each strand complements the other. PCR starts with the DNA sample, which is put in a reaction tube along with primers (short, synthetic pieces of single-stranded DNA that exactly match and flank the stretch of DNA to be amplified), deoxynucleotide triphosphates (dNTPs, the building blocks of DNA), buffers and a heat-resistant enzyme (polymerase). Heating the mixture separates the “template” strands of DNA. Then, at varying temperatures, the rest of the components in the mixture spontaneously organize themselves, building a new complementary strand for each original. At the end of each cycle the DNA count has doubled. If you start with one DNA molecule, at the end of 30 cycles (only a few hours later) there will be about a billion copies. Thus, if you are looking for a single gene among thou- sands, the game changes from “searching for a needle in a haystack” to “mak- ing a haystack of needles.” DUPLICATING DNA begins with a double-stranded stretch of DNA to be amplified, or copied (a). In a solution heated to 95 degrees Celsius (203 degrees Fahrenheit), hydro- gen bonds between the strands break, leaving two single strands (b). When the mixture is cooled to between 50 and 65 degrees C, specially manufactured DNA primers bind complementarily to each strand at points flanking the region to be copied (c). At 72 degrees C, polymerase enzymes extend the bound primers in one direction, using the original DNA as a template (d). The products are two new double strands of DNA, both identical to the original (e). This cyclic reaction takes only minutes or less and can be re- peated indefinitely. CRIME SCENE CLUES, such as a single hair or a drop of blood, can be analyzed using PCR. POLYMERASE ENZYME extends a bound primer. From the sur- rounding medium, it extracts a free-floating deoxynucleotide triphosphate (dNTP) that will complement the next unpaired position in the template strand of DNA. The enzyme then joins the dNTP to the end of the primer and moves on to the next position. EXPONENTIAL GROWTH of the DNA target occurs because the products of each cycle become the templates for the next cycle. STEVEN SENNE AP Photo ILLUSTRATIONS BY LAURIE GRACE

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TEMPLATE STRAND

dNTPPOLYMERASE

PRIMER

POLYMERASE

Working Knowledge112 Scientific American May 1998

W O R K I N G K N O W L E D G EPOLYMERASE CHAIN REACTION

by Elizabeth A. Dragon

Roche Molecular Systems

Polymerase chain reaction (PCR) is a technique that mimics nature’s way

of replicating DNA. First described in 1985, PCR has been adopted as

an essential research tool because it can take a minute sample of genetic

material and duplicate enough of it for study. PCR has been used to identify the

remains of Desert Storm casualties, to analyze prehistoric DNA, to diagnose

diseases and to help make identifications in police investigations.

DNA is most often found as a double-stranded molecule, twisted as a helix, in

which each strand complements the other. PCR starts with the DNA sample,

which is put in a reaction tube along with primers (short, synthetic pieces of sin-

gle-stranded DNA that exactly match and flank the stretch of DNA to be am-

plified), deoxynucleotide triphosphates (dNTPs, the building blocks of DNA),

buffers and a heat-resistant enzyme (polymerase). Heating the mixture separates

the “template” strands of DNA. Then, at varying temperatures, the rest of the

components in the mixture spontaneously organize themselves, building a new

complementary strand for each original.

At the end of each cycle the DNA count has doubled. If you start with one

DNA molecule, at the end of 30 cycles (only a few hours later) there will be

about a billion copies. Thus, if you are looking for a single gene among thou-

sands, the game changes from “searching for a needle in a haystack” to “mak-

ing a haystack of needles.”

DUPLICATING DNA begins with a double-stranded stretch of DNA to be amplified, orcopied (a). In a solution heated to 95 degrees Celsius (203 degrees Fahrenheit), hydro-gen bonds between the strands break, leaving two single strands (b). When the mix-ture is cooled to between 50 and 65 degrees C, specially manufactured DNA primersbind complementarily to each strand at points flanking the region to be copied (c). At72 degrees C, polymerase enzymes extend the bound primers in one direction, usingthe original DNA as a template (d). The products are two new doublestrands of DNA, both identical to the original (e). This cyclicreaction takes only minutes or less and can be re-peated indefinitely.

CRIME SCENE CLUES, such as a single hair ora drop of blood, can be analyzed using PCR.

POLYMERASE ENZYME extendsa bound primer. From the sur-rounding medium, it extracts afree-floating deoxynucleotidetriphosphate (dNTP) that willcomplement the next unpairedposition in the template strandof DNA. The enzyme then joinsthe dNTP to the end of the pri-mer and moves on to the nextposition.

EXPONENTIAL GROWTH of theDNA target occurs because the

products of each cycle become thetemplates for the next cycle.

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