DNA&RNA1b

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Polymerase Chain Reaction Fotocopiando o DNANational Center for Human Genome Research, National Institutes of Health. "New Tools for Tomorrow's Health Research."

Bethesda, MD: Department of Health and Human Services, 1992

Who would have thought a bacterium hanging out in a hot spring in Yellowstone National

Park would spark a revolutionary new laboratory technique? The polymerase chain reaction,

now widely used in research laboratories and doctor's offices, relies on the ability of DNA-

copying enzymes to remain stable at high temperatures. No problem for Thermus

aquaticus, the sultry bacterium from Yellowstone that now helps scientists produce millions

of copies of a single DNA segment in a matter of hours.In nature, most organisms copy their DNA in the same way. The PCR mimics this process,

only it does it in a test tube. When any cell divides, enzymes called polymerases make acopy of all the DNA in each chromosome. The first step in this process is to "unzip" the two

DNA chains of the double helix. As the two strands separate, DNA polymerase makes a

copy using each strand as a template.

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To copy DNA, polymerase requires two other components: a supply of the four nucleotide

bases and something called a primer. DNA polymerases, whether from humans, bacteria, or

viruses, cannot copy a chain of DNA without a short sequence of nucleotides to "prime" theprocess, or get it started. So the cell has another enzyme called a primase that actually

makes the first few nucleotides of the copy. This stretch of DNA is called a primer. Once the

primer is made, the polymerase can take over making the rest of the new chain.

A PCR vial contains all the necessary components for DNA duplication: a piece of DNA, large

quantities of the four nucleotides, large quantities of the primer sequence, and DNA

polymerase. The polymerase is the Taq polymerase, named for Thermus aquaticus, from

which it was isolated.

The three parts of the polymerase chain reaction are carried out in the same vial, but at

different temperatures. The first part of the process separates the two DNA chains in the

double helix. This is done simply by heating the vial to 90-75 degrees centigrade (about 165degrees Fahrenheit) for 30 seconds.

But the primers cannot bind to the DNA strands at such a high temperature, so the vial is

cooled to 55 degrees C. At this temperature, the primers bind or "anneal" to the ends of the

DNA strands. This takes about 20 seconds.The final step of the reaction is to make a complete copy of the templates. Since the Taq

polymerase works best at around 75 degrees C (the temperature of the hot springs where the

bacterium was discovered), the temperature of the vial is raised.The Taq polymerase begins adding nucleotides to the primer and eventually makes a

complementary copy of the template. If the template contains an A nucleotide, the enzyme

adds on a T nucleotide to the primer. If the template contains a G, it adds a C to the new

chain, and so on to the end of the DNA strand. This completes one PCR cycle.

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The three steps in the polymerase chain reaction - the separation of the strands, annealing

the primer to the template, and the synthesis of new strands - take less than two minutes.

Each is carried out in the same vial. At the end of a cycle, each piece of DNA in the vial has

been duplicated.But the cycle can be repeated 30 or more times. Each newly synthesized DNA piece can act

as a new template, so after 30 cycles, 1 billion copies of a single piece of DNA can beproduced! Taking into account the time it takes to change the temperature of the reaction

vial, 1 million copies can be ready in about three hours.

PCR is valuable to researchers because it allows them to multiply unique regions of DNA so

they can be detected in large genomes. Researchers in the Human Genome Project are

using PCR to look for markers in cloned DNA segments and to order DNA fragments in

libraries.

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Mutaes

Mutaes Pontuais: alterao de 1 s base pode alterar a protena

misssense: resulta na alterao de 1 aa

nonsense: resulta num codo stop

frame-shift: alterao "reading-frame" da mensagem gentica

Rearranjos cromosmicosEx: Bryce Bryer, "The Gene Boy" (Hippocrates Nov./Dec. 1989)

Mutaes nas sequncias de DNA geralmente ocorrem atravs de 1 de 2 processos:

1. danificao do DNA por agentes ambientais tais como luz u.v. (luz do sol), radiao nuclear ou certos produtos qumicos

2. erros que ocorrem quando 1 clula copia o seu DNA ao preparar-se para a diviso celular.

1. danificao do DNA por agentes ambientais

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Modificao das bases nucleotdicas

u.v., radiao nuclear e determinados produtos qumicospodem danificar o DNA pois alteram as bases nucleotdicasde modo a paerecerem outras bases nucleotdicas.

Quando as cadeias de DNA so separadas e copiadas, abase alterada emparelhar com 1 base incorrecta e causaa mutao. No exemplo 1 G "modificada" emparelhaagora com a T, em vez de formar o par normal com a C.

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Quebrando o esqueleto fosfato

Agentes ambientais tais como a radiao nuclear podem danificar o DNA pois quebram as ligaes entreoxignios (O) e grupos fosfato (P).

A quebra do esqueleto fosfato do DNA dentro do gene cria uma forma mutada do gene. possvelque o gene mutado produza uma protena com funes diferentes.Clulas com DNA quebrado tentaro resolver esta quebra juntando as extremidades livres a outrospedaos de DNA que existam dentro da clula. Isto cria um tipo de mutao chamada

Translocao. Se um ponto de translocao ocorre dentro ou nas proximidades de 1 gene, a suafuno pode ser afectada.

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2. erros criados durante a duplicao do DNAAntes da diviso celular, cada clula duplica a sequncia completa do DNA. A replicao do DNA comeaquando a protena DNA helicase separa a molcula de DNA em duas cadeias.

A seguir, 1 protena chamada DNA polimerase copia cada cadeia de DNA para criar 2molculas de duplacadeia de DNA.

Ocorrem mutaes quando a DNA polimerase faz 1 erro, o que acontece a cada 100,000,000 bases.Actualmente o n de erros que se mantm incoroprado no DNA menor que isto, uma vez que as clulascontm protenas reparadoras de DNA que corrigem muitos destes erros no DNA causados por mutagneos.As protenas reparadoras vem que nucletidos foram emparelhados incorrectamente e trocam a base erradapela certa.

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