Gene Location and Sequence

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Gene Location and Sequence

• Dr. Jason Linville
• University of Alabama at Birmingham
• jglinvil_at_uab.edu

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Summary

• Locating a gene
• Sequencing a gene
• Chain Terminating Nucleotides
• Degradation of DNA
• Cycle Sequencing

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Gene Location

• Facts

• We know how to clone a gene (cut up genome
  fragments into vectors)

• We know how to identify the gene we want (direct
  selection or hybridization probe)

It would be helpful to know where in the genome
the gene is located.
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Gene Location

• For small DNA molecules
• (plasmids phages)

If the total genome can be cut into a small
number of fragments, the fragment can be
identified through probing restriction fragments.
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Gene Location
Which of the 13 fragments contain the gene?
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Gene Location
For this to work, restriction map must be known.
(Figure 4.18 in book)
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Gene Location
Can also find location of gene within cloned
fragment.
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Gene Location

• Location on large DNA molecules

Restriction mapping of large genomes very time
consuming therefore, other methods used.
Step 1 Which chromosome?
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Gene Location

• Locating chromosome with gene

• Chromosomes can be separated by gel
  electrophoresis.

• Gene can be identified through southern
  hybridization.

Problem Chromosomes are too large for normal
electrophoresis.
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Gene Location
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Gene Location

• Orthogonal field alternation gel electrophoresis
• Results in separation of large chromosomes
• Still limited to smaller chromosomes

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Gene Location

• What about important animals you know, humans?

• Chromosomes way too large (gt50,000 kb) to be
  separated by gel electrophoresis

• Approximate gene location identified by in situ
  hybridization.

• If fluorescent label is used fluorescent in
  situ hybridization (FISH).

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Gene Location

• Cells fixed to slide ribonuclease added
  hydroxide denatures DNA

• Probe of gene shows location.

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DNA Sequencing
Two main techniques

• Chain-terminating nucleotides
• Also called Sanger-Coulson
• Can be coupled with PCR cycle sequencing

Chemical degradation (Maxam-Gilbert)
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DNA Sequencing

• Chain-terminating nucleotides

Molecule to be sequenced usually cloned into M13
molecule (single stranded DNA)
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DNA Sequencing

• Chain-terminating nucleotides

• Primer anneals to ss DNA adjacent to gene in
  vector (sequence known)
• Extension by DNA polymerase

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DNA Sequencing

• Chain-terminating nucleotides

Regular dNTPs (all) and one dideoxyNTP (ddNTP)
added.
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DNA Sequencing gt Chain Terminating

• Results in termination of product where an A
  occurs (when ddATP is added).
• Ending position varies because normal dATPs are
  also in the mixture.

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DNA Sequencing gt Chain Terminating

• 4 reactions carried out (each ddNTP)
• Separated on same gel

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DNA Sequencing gt Chain Terminating

• Thin acrylamide gel can separate 1 bp
  difference.
• Sequence read from bottom to top

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DNA Sequencing gt Chemical Degradation

• Chemical Degradation of DNA

Uses double stranded DNA M13 cloning not
necessary
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DNA Sequencing gt Chemical Degradation

• Chemical Degradation of DNA

• Several different ways to label and cleave DNA

• We will look at most common type.

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DNA Sequencing gt Chemical Degradation

• All identical restriction fragments
• Labeled with 32P heat DMSO denatured

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DNA Sequencing gt Chemical Degradation

• Separate strands with gel electrophoresis
• 4 digestions cleavage reagents/conditions allow
  only one breakage per strand

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DNA Sequencing

• Sequencing PCR Products

• One way is to separate strands after PCR
  (magnetic primer) then chain termination
• More commonly, cycle sequencing is used

Cycle sequencing uses principles similar to chain
termination reaction
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DNA Sequencing

• Thermal Cycle Sequencing

• Similar to PCR mixture
• Taq polymerase
• normal dNTPs

• Different from PCR mixture
• Only one primer added
• ddNTPs added

Not amplified, but
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DNA Sequencing
Two ways

• 4 separate reactions, each with one ddNTP

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DNA Sequencing
Two ways

• 4 separate reactions, each with one ddNTP

• Gel same as chain termination gel

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DNA Sequencing
Two ways

• Or, 4 differently labeled ddNTPs in the same tube
  (automated seq.)

• This is most common in forensic labs.

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DNA Sequencing
Real output from 310 Genetic Analyzer
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DNA Sequencing

• Long Sequences

• For long genes (gt750), gene can be digested and
  cloned sequenced
• Contig sequences aligned by identifying
  overlapping sequences

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DNA Sequencing
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DNA Sequencing
If fragment is too long, different restriction
enzyme can be used.