Chapter 19 - Chapter 19 Analyzing and Engineering Genes The...

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Chapter 19: Analyzing and Engineering Genes The importance of DNA technology o Automated Dideoxy Sequencing has allowed us to sequence the entire genome of many species including the human genome. The human genome project has sequenced all 3 billion nucleotides o We are now able to search our genome for the genes that cause genetic diseases. WE are working on strategies to replace these defective genes. o Knowing the DNA sequences of many genes for related species allows us to determine the phylogenetic relationships or history of these species o One of the greatest uses of DNA technology is to clone new genes into agricultural crops to increase yields, to increase disease resistance and resistance to herbicides. DNA purification o You need pure DNA to work with. Many different extraction protocols exist o Homogenize tissue in a buffer with proteinase k to degrade proteins o Small proteins are extracted with phenol. Lipids are extracted with chloroform. o Remaining solution contains DNA in solution o Precipitate DNA with 95% ETOH and low molarity salt solution o Centrifuge strands of DNA, was DNA pellet, and redissolve in distilled water. Cutting or Restricting the DNA o The nature and origin of restriction enzymes About 1000 restriction enzymes that recognize 150 recognition sites Proteins found in various species of bacteria used as mechanisms of defense against viruses o Most recognition sites are palindromes (KAYAK) o 4 base cutters cut the DNA more frequently than 6 base cutters because there’s more likely to see a 4 base pattern than a six base pattern o Different restriction enzymes cut the DNA in different ways resulting in blunt ends of sticky ends. These different types of ends are produced when the restriction enzymes cut in the same or different places on the two strands. o The overhangs of sticky ends are actually single stranded DNA that have a tendency to base pair with other compatible overhangs DNA Ligation (splicing) o DNA ligation involves joining pieces of DNA together o The process requires the enzyme DNA ligase
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o Ligations can join blunt end fragments or sticky end fragments. Sticky ends ligations are more efficient because of the natural attraction (hydrogen bonding) of the single stranded DNA o Sticky end ligations require fragments with compatible overhangs or
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Chapter 19 - Chapter 19 Analyzing and Engineering Genes The...

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