PowerPoint Slide Set to Accompany “Using M&M’s to Model Sanger’s Dideoxy DNA Sequencing Method” Joseph E. Conley, Alex J. Meisel, and James J. Smith Michigan State University Lyman Briggs College

DNA Sequencing – Maxam-Gilbert This was one of the first methods of DNA sequencing. This method allowed purified labelled DNA to be broken at specific bases by using specific reagents and conditions. The labelled fragments then would undergo electrophoresis in a polyacrylamide gel and be detected by autoradiography. It is important to note that this method called for the use of highly toxic chemicals (e.g., hydrazine), which limited its widespread use .

This figure from Maxam and Gilbert’s paper provides an example of a polyacrylamide gel used in Maxam-Gilbert Sequencing. The bands on the gel are arranged by the size of the DNA fragment. Shorter fragments are located at the bottom of the gel and longer fragments are located at the top.

DNA Sequencing – Sanger The DNA sequencing method developed by Sanger et al. (1977) is similar to the natural process of DNA replication. In 4 reaction tubes, a single template DNA strand is needed along with DNA polymerase, dNTPs, and a DNA primer. Then a specific ddNTP (ddNTP A, ddNTP C, ddNTP G, or ddNTP T) is added to each tube. The ddNTP’s are important because they terminate the extension of the growing DNA strand. Under specific conditions, new DNA strands of different lengths will be synthesized in each tube. These tubes are transferred to 4 lanes of an electrophoresis gel where DNA fragment length can be determined.

This figure from the paper by Sanger et al This figure from the paper by Sanger et al. shows an electrophoresis gel resulting from Sanger sequencing procedures.

Dideoxy Sequencing - The Sanger Method The Sanger Method was adapted preferably over the Maxam-Gilbert Method and ended up being used as the basis of the sequencing used for the Human Genome Project. Sanger’s gel was easier to read and Sanger’s method did not use the toxic chemicals that were needed in the Maxam-Gilbert Method.

Dideoxy Sequencing This is Figure 19-6a from Scott Freeman’s (2005) Biological Science textbook (2nd Ed.), Prentice-Hall Publishers, Upper Saddle River, NJ.

PCR as a reconstitution of DNA synthesis 8 B A C This slide is used to show the process of DNA replication and PCR. Note that key elements needed for PCR are DNA (Taq) Polymerase, a DNA template, a short primer sequence of DNA from which to extend nucleotide incorporation, and free dNTPs. DNA Polymerase plays a key role in DNA synthesis by catalyzing the addition of dNTPs to the growing strand. Sources of Illustrations: Part A - https://sites.google.com/site/thepandemicfluh1n1/9-influenza-viral-rna-synthesis/the-quasispecies-concept/10-the-error-prone-ways-of-rna-synthesis Part B - http://deenahere.hubpages.com/hub/PCR-stand-for-Polymerase-Chain-Reaction-Steps Part C - http://www.odec.ca/projects/2005/anna5m0/public_html/pcr.png

Dideoxy Sequencing: Laboratory Procedure Four reaction tubes, each with 32P labeled DNA primer AND one of each: dNTPs + ddNTP A dNTPs + ddNTP G dNTPs + ddNTP T dNTPs + ddNTP C

Dideoxy Sequencing: Radioactive Labels This is Figure 19-6b from Scott Freeman’s (2005) Biological Science textbook (2nd Ed.), Prentice-Hall Publishers, Upper Saddle River, NJ.

Dideoxy Sequencing: Radioactive Special type of gel made from polyacrylamide and urea Separates DNA strands that differ by as little as ONE NUCLEOTIDE Single lane on the gel for each reaction dNTPs + ddNTP A dNTPs + ddNTP G dNTPs + ddNTP T dNTPs + ddNTP C

Dideoxy Sequencing: Gel Apparatus

Dideoxy Sequencing: Radioactive Labels This is Figure 19-6c from Scott Freeman’s (2005) Biological Science textbook (2nd Ed.), Prentice-Hall Publishers, Upper Saddle River, NJ.

Dideoxy Sequencing with Fluorescent Dye (Hunkapiller, Hood et al Dideoxy Sequencing with Fluorescent Dye (Hunkapiller, Hood et al., Applied Biosystems)

Dideoxy Sequencing with Fluorescent Dye (Hunkapiller, Hood et al Dideoxy Sequencing with Fluorescent Dye (Hunkapiller, Hood et al., Applied Biosystems) Sequence data from the Genomics Core at the MSU Research Technology Support Facility (RTSF) Achieved using “Dye Terminators” ddNTP’s are labeled with a fluorescent dye Black, G; Blue, C; Green, A; Red, T

Dideoxy Sequencing with Fluorescent Dyes (Hunkapiller, Hood et al Dideoxy Sequencing with Fluorescent Dyes (Hunkapiller, Hood et al., Applied Biosystems) Each ddNTP labeled with a unique fluorescent dye; four sets of reactions pooled.