DNA technology

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  1. DNA sequencing: sanger method
    • Prepare four tubes with the same DNA as the template, all four dNTP: A, T, G, C, DNA polymerase, and a DNA primer.
    • Add concentration of a different ddNTP to each tube.
    • Four sets of complememntary sequences are generated from template DNA.
    • Separate the labled fragments by electrophoresis.
    • Go down lanes and list the bases in the order they are fragmented.
    • Use: for sequencing.
  2. DNA transformation:
    • Cloning vector is cleaved with restriction endonuclease.
    • DNA of interest is obtained by cleaving chromosome with RE.
    • Fragments are ligated to cloning vector.
    • DNA is introduced to host cell.
    • Propagation of transformed cell produces recombinant DNA.
    • Function: make plasmid copies.
  3. YAC and BAC:
    • YAC: yeast artificial chromisome. just a bigger cloning vector.
    • BAC: bacterial artificial chomosome. large plasmid construct.
  4. PCR:
    • Purpose: to replicate specific DNA sequences many times.
    • Heat to denature (separate strands).
    • Bind oligonucleotide primers to the specific sequence (need to know sequence).
    • Add heat stable DNA polymerase.
    • Duplicate the DNA sequence between the primers.
    • Repeat the above steps 20-30 times.
  5. Southern blot:
    • Function: show is DNA sequence is in sample.
    • Method:
    • (1) separate DNA segments by size in a gel (agarose gel)
    • (2) transfer separated segments to membrane
    • (3) identify segments. Cleave DNA molecule via restriction enzymes =>Agarose gel electrophoresis => fragments fractioned by size in gel => transfer to nitrocellulose filter (under denaturing conditions) => Nitrocellulose fiber with DNA fragments positioned identically to those in gel => hybridization with radioactively labeled DNA probe => X-ray film => radiograph showing hybrid DNA.
  6. DNA Mircroarry:
    • Purpose: Identifies genesequences in a whole genome or plasmid.
    • Allow DNA replication to begin at origin.
    • Break DNA into fragments, flurecently lable DNA.
    • Add labeled fragments into DNA microarray.
    • Allow complememntary base pairing to occur.
    • Examine microarray with flourencent scanner.
    • Microarrays:Identifies gene sequences in a whole genome or plasmid. It is quantitative, youcan tell how the intensity light changes over time (logarithmic orexponential). This technique can be used on any biomolecule: lipids, carbs,etc.. Each spot contains multiple exact copies of the same sequence, each spothas a unique sequence, and probes hybridize in a complementary fashion.
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DNA technology
2014-03-09 23:04:08
MCDB chapter

chapter 8-9
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