Introduction to Molecular Biology and Genomics

1
Introduction to Molecular Biology and Genomics

• Part One of a Short Course Series
• Functional Genomics and Computational Biology
• Lecture 6, November 9

2
Disease Gene Discovery

• Pedigree for familial forms
• Affected and Unaffected individuals
• single loci? Recessive/dominant?
• Search for linked marker from set of several
  thousand restriction site polymorphisms (RFLPs)

3
RFLPs

• 1/600,000 bp
• Type II enzymes have specific recognition sites
  (every 2-6Kbp), single base change results in no
  cutting
• No cutting results in change of size/number of
  fragment(s) two fragments become one
• Visualized by gel electrophoresis and
  hybridization specific to each marker
• Need several markers and many individuals to
  demonstrate distance

4
Cloning and Sequencing

• Assume tightly linked RFLP
• Identify clone in cosmid library for sequencing
  100,000 bp by chromosome walking (use new
  sequence to design primer for next sequence rxn
• Sequence both normal and disease
• Identify differences, correlate to pedigree
• Clone mRNA(s) from region for exon
  mappinggtgtgtsequence cDNAgtgtgtprotein

5
Postgenomic-era

• Assumptions
• Have entire genome
• Have a cDNA clones
• Have high resolution map with arrayed clones
  (parallel analysis)
• Have highly parallel gene expression analysis
• Have PCR-based technologies
• Have HT sequencing (384x2x600460,000 bases from
  a single machine in one day)
• Have computing power and bioinformatics

6
Two Prong Approach

• From the Genome
• From the Transcriptome/Proteome
• Differential Expression to get list of
  candidatesgtsequencegtimmediately get gene (from
  genome, remember) to check for concordance with
  pedigree data

7
From the Genome

• gt50,000 Sequence Tagged Sites (STS)
• Arrayed cosmid and YAC libraries
• Entire genome processed for exon/intron structure
  and ORFs
• Arbitrary PCR-based ID of genomic differences
  (AFLP)

8
From the Transcriptome/Proteome

• Differential Expression to get list of
  candidatesgtsequencegtimmediately get gene (from
  genome, remember) to check for concordance with
  pedigree data
• Microarrays or Beads and 2DGE-MS or CE-MS
• PCR-based differential analysis (DD-PCR or
  RNA-AFLP) gtgtgt sequence gtgtgt genome datagtgtgtpedigree
  concordance