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OCR A Biology

Dna Fragments Membrane Strands Polymerase Lots Step Temp

Producing a DNA profile involves DNA extraction using PCR, digestion with restriction enzymes, separation of fragments via electrophoresis, and transfer to a membrane through Southern blotting for hybridization with probes and subsequent visualization.

Producir un perfil de ADN implica extraer ADN mediante PCR, digerirlo con enzimas de restricción, separar los fragmentos por electroforesis y transferirlos a una membrana mediante Southern blotting para su hibridación con sondas y visualización posterior.

Front Producing DNA profile
Back
  • Extracting DNA using Polymerase chain reaction (PCR) from small tissue sample to form lots of DNA; the DNA sample, XS nucleotide bases, small primer DNA bases and DNA polymerase mixed in vial and placed into PCR machine and process forms lots of copies of DNA; step 1- temp at 90C to dentaure DNA by breaking H2 bonds so strands seperate, step 2 - temp dec to 60C and primers bind to DNA bases, step 3 - temp inc to 75C, where DNA polymerase works optimally to attach primers to nucleotide bases to form complementary strands, cycle keeps repeating until lots formed
  • Digesting sample - strands of DNA cut using restriction endonucleases at specific site (restriction site), mixture of restriction enzymes used at introns to ensure micro/mini satelites intact
  • Seperating DNA fragments - electrophoresis where -ve charged DNA moves, when current applied, to positive end of agarose gel, rate of movement depends on mass/length of fragments (smaller fragments travel faster and further), gel placed in alkaline buffer to denature fragments and DNA strands of each fragment seperate, then during southern blotting, strands transferred to nylon membrane (which is placed over gel), membrane draws the alkali sol (with DNA), so single strand DNA fragments transferred to membrane at same relative positions as in gel
  • Hybridisation - XS radioactive/florescent DNA probes (DNA/RNA complementary to bases) added to fragments on membrane where they bind
  • Evidence - X-rays of membrane taken of radioactive, UV light shows up florescent tags if flourescent probes used

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