Recombinant DNA and Gene expression

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Recombinant DNA and Gene expression
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The reproductive cycle of HIV, a retrovirus
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The lytic and lysogenic cycles of phage ?, a
temperate phage
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Conjugation and recombination in E. coli
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A process starts with breakage at the
corresponding points of the homologous strands of
two paired DNA duplexes. The breakage allows
movement of the free ends created by the nicks.
Each strand leaves its partner and crosses over
to pair with its complement in the other duplex.
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Recombination is initiated by a double-strand
break, followed by formation of single-stranded
3 ends, one of which migrates to a homologous
duplex. A break must be made in one duplex in
order to generate a point from which single
strands can unwind to participate in genetic
exchange. Both strands of a duplex must be
broken to accomplish a genetic exchange.
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Overview of gene cloning with a bacterial plasmid
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Using restriction enzymes to make recombinant DNA

• Restriction enzymes Gene cloning and genetic
  engineering were made possible by the discovery
  of enzymes that cut DNA molecules at limited
  number of specific location.
• Restriction sites Hundreds of different
  restriction enzymes have been identified. Each
  enzyme is very specific, recognizing a particular
  short DNA sequence at specific points within the
  restriction site.

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Genomic libraries
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Nucleotides
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Nucleic Acids
Phosphodiester bonds
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Whole-genome shotgun approach to sequencing
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Microarray Technology

• Microarray technology is emerging as a powerful
  platform of biological exploration.
• The technology are provided by some ideas for the
  standardization of experimental design,
  microarray formats, detection systems, data
  outputs, and software tools.

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Biological questions and the microarray life cycle

• Gene expression in biological processing in cell
• Gene expression between different biosystems,
  animals, even organs
• Gene expression with dynamics and dependence
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The processing of the microarray experiment

• Sample preparation, mRNA amplification and
  labeling
• Hybridization, image scanning and digitization
• Data analysis and mining

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Sample preparation, mRNA amplification and
labeling
Sample preparation
Microarray assays generate a large data flow,
placing a premium on high-quality nucleic acid
samples.
RNA degradation
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Sample preparation, mRNA amplification and
labeling
mRNA amplification and labeling

• T7-based RNA amplification scheme
• mRNA reverse transcription with primer conjugated
  T7 promoter
• Amplification RNA and labeling with T7 polymerase

Comparing DNA amplification with PCR method,
T7-based RNA amplification scheme is a linear
process, skewing of resultant amplified RNA
population relative to the input mRNA is
minimized.
Output garbage
Input dust
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Microarray Tools and Technologies
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Two color comparative micoarray
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Hybridization, image scanning and digitization
Hybridization

• Hybridization buffer condition
• Sample concentration
• Temperature

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Hybridization, image scanning and digitization
Image scanning and digitization
Data quantification and quality measurement
Preobjective scanning
Charge-coupled device
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Data analysis and mining

• Normalization and transformation
• Data visualization
• Cluster analysis

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Gene therapy using a retroviral vector