Power Point Slide Set to Accompany Using MMs

  • Slides: 16
Download presentation
Power. Point Slide Set to Accompany “Using M&M’s to Model Sanger’s Dideoxy DNA Sequencing

Power. Point 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 1

2 DNA Sequencing – Maxam-Gilbert

2 DNA Sequencing – Maxam-Gilbert

3

3

DNA Sequencing – Sanger 4

DNA Sequencing – Sanger 4

5

5

Dideoxy Sequencing The Sanger Method 6

Dideoxy Sequencing The Sanger Method 6

7 Dideoxy Sequencing

7 Dideoxy Sequencing

PCR as a reconstitution of DNA synthesis 8 B A C 8

PCR as a reconstitution of DNA synthesis 8 B A C 8

9 Dideoxy Sequencing: Laboratory Procedure n Four reaction tubes, each with 32 P labeled

9 Dideoxy Sequencing: Laboratory Procedure n Four reaction tubes, each with 32 P labeled DNA primer AND one of each: • d. NTPs + dd. NTP A • d. NTPs + dd. NTP G • d. NTPs + dd. NTP T • d. NTPs + dd. NTP C

Dideoxy Sequencing: Radioactive Labels 10

Dideoxy Sequencing: Radioactive Labels 10

11 Dideoxy Sequencing: Radioactive n n n Special type of gel made from polyacrylamide

11 Dideoxy Sequencing: Radioactive n n n 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 • d. NTPs + dd. NTP A • d. NTPs + dd. NTP G • d. NTPs + dd. NTP T • d. NTPs + dd. NTP C

12 Dideoxy Sequencing: Gel Apparatus

12 Dideoxy Sequencing: Gel Apparatus

Dideoxy Sequencing: Radioactive Labels 13

Dideoxy Sequencing: Radioactive Labels 13

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

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

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

Dideoxy Sequencing with Fluorescent Dye (Hunkapiller, Hood et al. , Applied Biosystems) n n Sequence data from the Genomics Core at the MSU Research Technology Support Facility (RTSF) Achieved using “Dye Terminators” • dd. NTP’s are labeled with a fluorescent dye • Black, G; Blue, C; Green, A; Red, T 15

16 Dideoxy Sequencing with Fluorescent Dyes (Hunkapiller, Hood et al. , Applied Biosystems) Each

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