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Department of Biological and Environmental Sciences
Genetics
 Dr. David A. Johnson
Biol 333

Gene Technology and Genomics :-)
pp. 546-549, 554-557, 559-565, 586-588

  • Gene Technology: The era of gene technology began in the 1970s with the development of several methodologies such as the use of restriction enzymes (restriction endonucleases), in vivo gene cloning, and DNA sequencing.
    • Restriction Enzymes: These endonucleases recognize a certain palindromic DNA sequences and cut the molecule there.
      • Summary: These are endonucleases that  recognize a certain palindromic DNA sequences and cut the molecule there.


    • Gel Electrophoresis: This technique can be used to separate DNA molecules according to size. (Know: Southern blot, Northern blot, Western blot -- pp. 560-561)
      • Summary: This technique separates DNA molecules according to size. (Know: Southern blot, Northern blot, Western blot -- pp. 560-561)
    • Shotgun and Other Cloning Methodologies: Early gene cloning experiments were done using restriction enzymes to ligate target DNA to vector DNA. This hybrid molecule was then used to transform E. coli. Cloning procedure like this can be used to build genomic libraries or cDNA libraries. However, mRNA is isolated and reverse transcriptase is used in building cDNA libraries.
      • Summary: Early gene cloning experiments placed a random foreign DNA segment in a plasmid then placed that plasmid in E. coli. Subsequent cloning experiments placed a DNA copy of eukaryotic mRNA (cDNA)(the gene minus its introns) into E. coli. This methodology led to creating bacteria that could produce human proteins, like insulin.

    • PCR: This in vitro methodology amplifies a segment of DNA 230 fold.
      • Summary: PCR is a inexpensive in vitro DNA cloning technique that greatly amplifies a specific DNA segment. It can start with a minute quantity of target DNA.
  
    • DNA Sequencing: Early sequencing methods used radioactive labels. The late 20th century's technology was Sanger sequencing. The technology of  uses the dideoxy method. "Next-Generation Sequencing" is quickly becoming more affordable and is replacing Sanger sequencing. Some of these methodologies include Pyro Sequencing and HiSeq/miSeq.
      • Summary: The Sanger sequencing method determines the sequence of a segment of DNA a few hundred up to a thousand bp in length. Next-generation sequencing greatly increases the quantity of DNA that can be sequenced.

    • The Human Genome Project: The mega-sequencing project undertaken at the end of last century had as its goal the sequencing of the 3 billion base-pair human genome. (What are SNPs?)
      • Summary: The HGP sequenced of the 3 billion base-pair human genome.
    • Microarrays (Gene Chips): One of the many methodologies used in genomics is the microarray. With this technique, it is possible to ask questions like, "What genes are expressed in cancer cells that are not expressed in normal cells."
      • Summary: Microarrays rely on hybridization of DNA or RNA extracted from an organism to DNA molecules that are fixed on a gene chip. One use of microarrays is to determine which genes, out of the 20000+ genes, are transcribed in a specific human tissue.
    • Gene Knockdown Using RNAi: RNA interference (RNAi) (see Gene Regulation outline) can suppress the translation of one specific mRNA. Therefore it can be used to study what the effect of reducing the expression of a specific gene is--thereby elucidating the function of that gene.
      • Summary: Using RNAi, gene knockdown can turn down or turn off the expression of a specific gene, giving hints to the genes functions.
    • CRISPR/Cas9: What is it? (Be able to answer this question on the next test.) See this link and this link and this link.
      • Summary: from your paper.
    • Genomics:
      • Summary: Genomics is the study of the entire genome and its effect on a trait, cell, organism. (Gene knockdown is one practical genomics methodology.)