Text Version of PCR Slide Show

• THE FOLLOWING PAGES DESCRIBE THE POLYMERASE CHAIN REACTION, A PROCEDURE FOR AMPLIFYING A SPECIFIC FRAGMENT OF DNA.
• DIAGRAM S ARE USED TO HELP VISUALIZE STEPS IN THE PCR PROCESS.
• DESCRIPTIONS OF THE PROCESS ARE GIVEN AS TEXT BELOW EACH DIAGRAM.

• THERE ARE MILLIONS OF DIFFERENT GENES OR SEQUENCES WITHIN ANY DNA SAMPLE (BLOOD, TISSUE, ETC.).
• A SPECIFIC SEQU ENCE IS SELECTED TO BE AMPLIFIED (RED ABOVE). THIS SEQUENCE CAN BE ANY GENE OF INTEREST OR A NON-CODING AREA OF DNA.

• IN ORDER TO COPY THAT GENE, A SHORT SEQUENCE ON EITHER SIDE OF THE SECTION MUST BE KNOWN. THIS REGION (BLUE ABOVE) WILL S ERVE AS A PRIMER ATTACHMENT SITE TO COPY THE DNA SEGMENT.

• IN ORDER TO AMPLIFY A SPECIFIC FRAGMENT OF DNA, SEVERAL THINGS ARE NEEDED, INCLUDING PRIMERS AND DNA POLYMERASE, AN ENZYME WHICH COPIES DNA.
• PRIMERS ARE SHORT PIECES OF DNA OR R NA DESIGNED TO PAIR WITH GENOMIC DNA AT A SPECIFIC ATTACHMENT SITE FOR THE MAIN PURPOSE OF HELPING THE DNA POLYMERASE BIND AT THE DESIRED SECTION.

• WITHOUT A SHORT PIECE OF DNA(OR RNA) TO ATTACH TO, DNA POLYMERASE CANNOT COPY A DNA STRAND. < /ul>
  • NUCLEOSIDE TRIPHOSPHATES, THE BUILDING BLOCKS OF DNA ARE ALSO NEEDED. EACH NUCLEOSIDE TRIPHOSPHATE CONSISTS OF:
    • A BASE (ADENINE, THYMINE, CYTOSINE OR GUANINE).
    • A SUGAR AND THREE PHOSPHATES.
  • PCR REQUIRES SEVE RAL CYCLES OF AMPLIFICATION. EACH CYCLE CONSISTS OF THREE TEMPERATURE CHANGES.
    • THE STARTING TEMPERATURE (95 C) SEPARATES THE DNA STRANDS.
    • A LOWERED TEMPERATURE (50-60 C) ALLOWS PRIMERS TO BIND TO COMPLEMENTARY SEQUENCES IN THE DNA.
    • A S LIGHTLY HIGHER TEMPERATURE (72 C) ALLOWS DNA POLYMERASE TO ATTACH TO THE PRIMERS AND COPY THE DNA STRANDS (EXTENSION).
  • DNA STRANDS ARE SEPARATED BY HEATING @ 94 C.
  • THE TEMPERATURE IS LOWERED TO 54 C TO ALLOW PRIMERS TO PAIR WITH COMPLEMENTARY DNA SEQUENCES.
  • MAKING NEW DNA MOLECULES:
    • DNA POLYMERASE ATTACHES TO THE PRIMERS @ 72 C.
    • DNA POLYMERASE ADDS NUCLEOSIDE TRIPHOSPHATES TO THE PRIMERS TO COPY THE DNA STRANDS.
  • COPYING I S COMPLETED FOR EACH STRAND.
  • THE PROCESS IS REPEATED IN THE NEXT CYCLE. THE TEMPERATURE IS RAISED AGAIN TO SEPARATE THE DNA STRANDS.
  • THE TEMPERATURE IS LOWERED TO ALLOW PRIMERS TO ANNEAL. DNA POLYMERASE ATTACHES TO THE PRI MERS AND DNA IS COPIED TO MAKE 4 STRANDS OF DNA.
  • THE PROCESS OF COPYING DNA STRANDS IS REPEATED 32-35 TIMES. WITH EACH AMPLIFICATION CYCLE, THE NUMBER OF COPIES OF THE DNA SEQUENCE IS DOUBLED UNTIL MILLIONS OF COPIES HAVE BEEN MADE.
  • THE PCR PRODUCT IS RUN ON AN 0.8% AGAROSE GEL TO ENSURE THAT AMPLIFICATION OCCURRED. PHI X CUT WITH THE RESTRICTION ENZYME HAE III, IS RUN ON THE GEL AS A MOLECULAR WEIGHT MARKER.
  
  
  
  

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