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Lecture 5

BIL 250 Lecture Notes - Lecture 5: Electrophoretic Mobility Shift Assay, Human Genome Project, Dna Sequencing


Department
Biology
Course Code
BIL 250
Professor
karenalvarezdelfin
Lecture
5

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DNA Sequencing is the Ultimate Way to Characterize DNA Structure at
the Molecular Level
Dideoxynucleotide (ddNTP) chain termination sequencing (Sanger)
Most common method of DNA sequencing
Dideoxynucleotide: deoxynucleotide with a hydrogen at 3’ instead of
an OH
Dideoxynucleotide causes DNA synthesis to terminate when get
randomly incorporated into the polynucleotide chain
Computer Automated DNA Sequencing
Since early 1990s, DNA sequencing has been done through computer
automated Sanger reaction based technology
Generates large amounts of sequence DNA
Enabled rapid progress of Human Genome Project
Detecting the Binding of Proteins to DNA
This procedure can determine if a protein or mixture of proteins is
capable of binding to a given DNA or RNA sequence
Study of DNA protein interactions useful for understanding
transcription factor activity and histone placement
Gel retardation assay
Also called gel mobility shift assay or electrophoretic mobility shift
assay
Binding of a protein to a fragment of DNA retards its rate of movement
through a gel
Must be performed under non-denaturing conditions as unfolding of
protein or separation of DNA strands would cause it to release DNA
protein interaction
Complexes are electrophoresed through a gel
Complexes that migrate more slowly indicate that DNA was bound by
protein
Lower mass and therefore fast migration
Higher mass and therefore slow migration
An antibody that recognizes the protein can be added ot this mixture
to create an even larger complex with a greater shift
This method is referred to as a supershift assay, and is used to
unambiguously identify a protein present in the protein nucleic acid
complex
CRISPR-Cas9 Gene Editing Technology: creating knockout and
transgenic organisms for studying Gene Funciton
CRISPRs (clustered regularly interspaced palindromic repeats) are a
series of short direct repeats interspaced with short sequences in the
genome in numerous bacteria and archaea
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