MBIO 3410 lecture Nov 22 2007

MBIO 3410 lecture Nov 22 2007 - DNA Sequencing 1 DNA...

Info iconThis preview shows pages 1–7. Sign up to view the full content.

View Full Document Right Arrow Icon
1 DNA Sequencing
Background image of page 1

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

View Full DocumentRight Arrow Icon
2 DNA Sequencing Each of the two main methods of sequencing long DNA molecules (chemical and enzymatic) involves the production of a set of different sized molecules with one common end which are then separated by polyacrylamide gel electrophoresis (PAGE) to allow reading of the sequence The two methods are: The chemical method of Maxam and Gilbert The enzymatic Sanger method
Background image of page 2
3 The Chemical Method of Maxam and Gilbert This method requires that the DNA fragment is labeled at one end Usually by adding either a radioactive phosphate to the 5’- or 3’-end or a nucleotide to the 3’-end This method involves base specific cleavages which occur in two steps: 1. The base is 1 st modified using chemicals Dimethyl sulfate (DMS) methylates G bases Formic acid will attack the purines A and G Hydrazine is used to hydrolyze at pyrimidines (C + T) but high salt inhibits the T reaction Modification of T with potassium permanganate 2. Followed by piperdine cleavage of the sugar phosphate backbone of the DNA at that site
Background image of page 3

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

View Full DocumentRight Arrow Icon
4 Limiting incubation times or concentrations of components in the base modifying reactions ensures a ladder of progressively longer molecules is created rather than complete cleavage to short nucleotides Four lanes on a sequencing gel (G, A+G, C+T, C) allow the sequence to be determined Problems associated with this technique: Limited by length of fragment sequenced (~100 bp max) Use of harsh chemicals
Background image of page 4
5 Sanger Method This method uses four specific 2’, 3’dideoxynucleotides (ddNTPs) to terminate enzymatically synthesized copies of the template A sequencing primer is annealed to a ssDNA template molecule and a DNA polymerase extends the primers using dNTPs The extension reaction is split into four and each quarter is terminated separately with one of the four specific ddNTPs and the four samples (usually radioactive) are analyzed by PAGE The dideoxynucleotides act as chain terminators since they have no 3’-OH group on the deoxyribose which is needed by the polymerase to extend the growing chain
Background image of page 5

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

View Full DocumentRight Arrow Icon
6 The sequencing reaction is split into four separate parts To each was added a mixture of the four nucleotide triphosphates (dNTPs) required for the synthesis of new DNA One of these was radio-labeled so that the newly synthesized DNA could be easily detected A single dideoxynucleotide triphosphate (either ddATP, ddGTP, ddCTP or ddTTP) was included in each reaction at a concentration of approximately 1/10 of its deoxynucleotide counterparts e.g. In the reaction containing ddATP when a T residue occurs on the template strand, in most cases a dATP will be inserted into the newly synthesized chain At a relatively low frequency the dideoxy form of the nucleotide will be incorporated and the chain will terminate at this point Since many DNA molecules are produced at the same time, this process results in the formation of a population of partially
Background image of page 6
Image of page 7
This is the end of the preview. Sign up to access the rest of the document.

Page1 / 27

MBIO 3410 lecture Nov 22 2007 - DNA Sequencing 1 DNA...

This preview shows document pages 1 - 7. Sign up to view the full document.

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