lecture13

lecture13 - 3D Geometry of the Human Genome Background...

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Unformatted text preview: 3D Geometry of the Human Genome Background Lieberman-Aiden et al. (2009) Comprehensive Mapping of Long-Range Interactions Reveals Folding Principles of the Human Genome. Science 326: 289-293. Background: Understanding the 3D conformation of the genome can provide insight into chromatin structure, gene expression, and cellular processes. Imaging of painted chromosomes is laborious and only provides information about the spatial positions of a small number of markers at the time of imaging. Chromosome conformation capture (3C) can be used to identify pairs of loci that come into close contact, but is also locus-specific. This study introduces a new genome-wide version of 3C called Hi-C. Jay Taylor (ASU) APM 530 - Lecture 13 Fall 2010 1 / 19 3D Geometry of the Human Genome Background Hi-C consists of six steps. Step 1: The DNA is crosslinked. Formaldehyde ( CH 2 O ) forms crosslinks between nucleic acids and proteins. Crosslinking mainly occurs between amino groups and nearby nitrogen atoms. Jay Taylor (ASU) APM 530 - Lecture 13 Fall 2010 2 / 19 3D Geometry of the Human Genome Background Step 2: The crosslinked DNA is digested. Restriction endonucleases (REs) are bacterial enzymes that cut DNA at specific nucleotide sequences. This study used HindIII and NcoI which recognize the palindromic sequences 5-A A G C T T-3 and 5-C C A T G G-3 . The human genome contains approximately 850,000 HindIII restriction sites. HindIII and NcoI both create DNA fragments with complementary 5 overhangs known as sticky ends . Jay Taylor (ASU) APM 530 - Lecture 13 Fall 2010 3 / 19 3D Geometry of the Human Genome Background Step 3: The sticky ends are filled and marked with biotin. The reactive sticky ends are converted to less reactive blunt ends by the addition of nucleotides and DNA polymerase. The blunt ends are also marked with biotin (vitamin B7), which is incorporated as biotin-dCTP. Jay Taylor (ASU) APM 530 - Lecture 13 Fall 2010 4 / 19 3D Geometry of the Human Genome Background Step 4: The filled DNA ends are ligated. Ligases are enzymes that join dsDNA strands together. T4 DNA ligase is specifically used to anneal blunt ends. By carrying out the reaction under very dilute conditions, ligation mainly occurs between the crosslinked DNA fragments....
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This note was uploaded on 03/11/2012 for the course APM 530 taught by Professor Staff during the Fall '10 term at ASU.

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lecture13 - 3D Geometry of the Human Genome Background...

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