chapter12

chapter12 - BCH 4053 Summer 2001 Chapter 12 Lecture Notes...

Info iconThis preview shows pages 1–4. Sign up to view the full content.

View Full Document Right Arrow Icon
Chapter 12, page 1 BCH 4053 Summer 2001 Chapter 12 Lecture Notes Slide 1 Chapter 12 Structure of Nucleic Acids Slide 2 Primary Structure of Nucleic Acids • Ability to sequence DNA required discovery of restriction enzymes, which could produce homogeneous fragments • (pure DNA, being very large, will shear randomly to make a mixture of fragments) • Sequencing involves labeling nucleotides with a radioactive tag ( 32 P) or a flourescent tag so that small amounts can be detected Slide 3 Sequencing Strategies • Enzymatic—uses DNA polymerase and dideoxy nucleosides as chain terminating reagents • Developed by F. Sanger—earned him a second Nobel Prize • Chemical—uses base-specific chemical cleavage reactions developed by Maxam and Gilbert
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full Document Right Arrow Icon
Chapter 12, page 2 Slide 4 Sanger Method • Uses DNA polymerase, which requires a primer , a template , and all deoxynucleoside triphosphates • (See diagram Figure 12.2) • Inclusion of a small amount of a dideoxynucleoside triphosphate produces random fragments with no free 3’-OH, so chain terminates • Run four separate incubations each containing one dideoxynucleotide (ddATP, ddGTP, ddCTP or ddTTP). Slide 5 Sanger Method, con’t. • Because ddNTP concentration is low, most of the time the normal dNTP is incorporated. • Occasionally a ddNTP is inserted, and the chain terminates. • A set of “nested fragments” is produced. Slide 6 Nested Fragments from Chain Termination • For example, if the DNA sequence were: ATCCGGTAGCAATCGA • Termination at G would produce ATCCG ATCCGG ATCCGGTAG ATCCGGTAGCAATCG • These fragments can be separated by size on electrophoresis (See Figure 12.3) The fragments must be labeled some way so they can be detected. One technique is to use one of the dNTP’s labeled with 32 P. Another is to put a flourescent label onto one of the nucleotides, or to attach a flourescent label to the primer oligonucleotide.
Background image of page 2
Chapter 12, page 3 Slide 7 Maxam-Gilbert (Chemical Cleavage) Method • Developed before Sanger’s method, but not as widely used now. • Nested fragments are produced by specific chemical cleavage. • DNA labeled with 32 P, usually at the 5’ end • Reaction conditions set so that on average only one cleavage will occur in a single molecule. Slide
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full Document Right Arrow Icon
Image of page 4
This is the end of the preview. Sign up to access the rest of the document.

{[ snackBarMessage ]}

Page1 / 8

chapter12 - BCH 4053 Summer 2001 Chapter 12 Lecture Notes...

This preview shows document pages 1 - 4. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online