Revolutionary technology

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Give me a long enough lever and I will move the
universe

• Who said it?

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Biochemistry 441Lecture 3Ted YoungJanuary 11,
2008

• DNA-based information technologies
• Reading Ch 9.1-9.3 and related problems in the
  book.

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Revolutionary technologies

• 1. Restriction enzymes
• 2. Cloning
• 3. DNA sequencing
• 4. PCR(Polymerase chain reaction)
• 5. SNPs and RFLPs
• 6. Oligonucleotide mutagenesis
• 7. CHIPs (DNA arrays)
• ((8. Chemical synthesis of DNA
  oligonucleotides- will not be covered))

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Revolutionary DNA technologies

• Most new technologies require or lead to the
  ability to determine and manipulate the sequence
  of DNA.
• What can we learn from the DNA sequence?
• (see PLOS Biology (2003)1156 Comparative
  Genomics http//biology.plosjournals.org).

1. Evolutionary implications relatedness of
organisms, relatedness of individuals of the
same species inherited mutations
mutations associated with human disease
2. Technology for gene analysis and
manipulation-knocking-out genes to determine
their role in physiology, metabolism, and
development
3. Determination of protein primary structure
protein function (if homologous to a protein of
known function) allows 3D structure prediction.
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Determination of DNA sequence

• The Classical method purifying a collection of
  identical DNA molecules.
• Marking or labeling a single end (end A)
• Separating molecules which differ by a single
  nucleotide and determining the base at the
  opposite end (end B).

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The first problem homogeneous DNA

• DNA molecules can be huge
• Small virus5000-50000bp

Human genome 3,000,000,000 bp ???????? meters
(3000 mega base pairs 3 million kilobp).
Drosophila chromosome 1.2 cm ??????? bp
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The second problem specific fragmentation

• How can small DNA fragments with a unique
  sequence be obtained?

(Hint-how was this problem solved in protein
sequencing?)
Single chromosome
Mechanical shear breaks the DNA into
random-size/sequence pieces
etc
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Restriction endonucleases

• Discovery Host restriction-modification in
  bacteria.
• Type II restriction enzymes separate
  endonuclease and methylase activities.
• Nomenclature E. coliEco, etc

Palindromic symmetry Able was I ere I saw
Elba Sex at noon taxes Eve is a sieve A Santa
at NASA, etc (Also called inverted repeats.)
Type II restriction endonucleases recognize and
cut the same sequence
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Structure of a restriction endonuclease

• Restriction enzymes are usually dimers of
  identical subunits, analogous to the symmetry of
  their binding sites in DNA.

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Molecular cloning using bacterial plasmids

• Identification and characterization of bacterial
  plasmids was an essential part of the
  development of modern DNA technology

AmpicillinR
Gene encoding drug-resistance
Sequence allowing replication in E. coli (ori,
origin of replication)
Unique restriction sites
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Creating a recombinant DNA molecule (cloning)
involves four steps
Tet gene
Amp gene
1.
1. Cutting vector and insert DNA with
compatible restriction enzymes. 2. Annealing
the sticky ends. 3. Covalently sealing the
annealed ends with DNA ligase ATP. 4.
Transformation of competent bacteria, selecting
for eg Ampicillin-resistance
2. and 3.
4.
Select tetR
AmpS tetS-E. coli
Screen for amp
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Identifying clones
B. Gene- specific identi- fication- Colony hybridi
- zation.
A. General (not gene-specific)
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Restriction enzyme maps

• Cleavage of DNA with restriction enzymes provides
  landmarks and sequence information.
• How frequently are specific sites found?
• How many sites are expected? Does this change if
  the DNA is circular?

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Automated DNAsequencing allowed the completion
of the human genome sequence faster than expected
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Genomic sequencing-the end game
Using robotics, capillary electrophoresis, tens
of automated sequencers, and computer reading and
analysis of the output megabp of sequence can be
obtained per day.
The major rate limiting steps are 1.putting the
random fragments together, ie re-establishing
the correct order (repeated sequences are a
major problem)
2. Assuring that no pieces are missing 3. Finding
genes-based on start and stop signals intron-exo
n sequences open reading frames (ORFs). This is
all part of the Annotation that makes sense out
of the information. 4. Finally-what is the
function of the newly-discovered genes?
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Comparative genome sizes-completed genome projects
Organism size( X106bp) genes
gene density Human(all mammals) 3000
30,000 1 gene per 100,000 bases Zebrafish 1750
? ? Drosophila (fruit fly) 135.6 13,061 1 gene
per 13,781 bases Arabidopsis (plant) 100
25,000 1 gene per 4000 bases C. elegans
(roundworm) 97 19,099 1 gene per 5079
bases S. cerevisiae (yeast) 12.1 6034 1 gene
per 2005 bases E. coli (bacteria) 4.67 3237 1
gene per 1443 bases H. influenzae (bacteria) 1.8
1740 1 gene per 1034 bases Gene density has
changed dramatically during evolution. What is,
or is there, a function for all of the non-coding
DNA?
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The human (and other) genomes-what have we
learned?

• Number of human genes is fewer than expected (but
  alternative slicing increases that number)
• Gene arrangement is often conserved between
  species.
• Polymorphisms, differences between individuals in
  the same species, are very frequent (1 change
  per 1000 nucleotides).
• The number of gene families is similar in flies
  and humans.
• Most human genes have orthologues in other
  species-even down to yeast. This fact is
  important for studying gene function.

www.wellcome.ac.uk/en/genome/genesandbody
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New methods of sequencingShotgun cloning and
sequencing

• A procedure developed to skip the laborious and
  time consuming process of identifying and mapping
  specific DNA fragments. Instead, random fragments
  are cloned and sequenced. Their order is
  determined solely from the sequence of
  overlapping DNA fragments.