Cycle Sequencing

Broad and Long Term Objective To characterize a single clone from an Emiliania huxleyi cDNA library using sequence analysis To characterize a single clone from an Emiliania huxleyi cDNA library using sequence analysis

Research Plan Preparation of Competent Cells and Bacterial Transformation Growth of Transformant and Plasmid MiniPrep Cycle Sequencing Sequence analysis

Today’s Laboratory Objectives 1.To Sequence the E. huxleyi cDNA insert in the pMAB plasmid using cycle sequencing using cycle sequencing 2.To learn how to interpret cycle sequencing data 3.To learn how to characterize a DNA sequence using various web-based bioinformatics tools

What is Cycle Sequencing? Based on the Sanger Dideoxy chain termination method Based on the Sanger Dideoxy chain termination method DNA synthesis reaction whereby fluorescent dideoxynucleotides are incorportated into the newly replicated DNA by DNA polymerase in a primer extension reaction DNA synthesis reaction whereby fluorescent dideoxynucleotides are incorportated into the newly replicated DNA by DNA polymerase in a primer extension reaction Thermal cycling reaction Thermal cycling reaction

Primer Extension Reaction Whenever a fluorescently labeled dideoxynucleotide is incorporated chain termination occurs DNA Sequencing: Dideoxy Chain Termination Method

DNA Polymerase 1.an enzyme used in sequencing to extends the 3' end of a primer along a ssDNA template using dNTPs and ddNTPs 1.DNA pol-1 cannot distinguish a between normal dideoxynucleotides (ddNTPs) and a chemically modified fluorescent ddNTPs 3.Whether the enzyme incorporates by completmentary base pairing a dNTP or a fluorescent ddNTP depending on the concentration ratio of ddNTPs/dNTPs 4. Each time the enzyme place a ddNTP the sequence will be "terminated", because ddNTPs don't have a 3' end.

Primer Extension Reaction Components DNA template DNA template Primer Primer Fluorescently labeled ddNTPs Fluorescently labeled ddNTPs dNTPs dNTPs Buffer Buffer Amplitaq= termal stable DNA Polymerase Amplitaq= termal stable DNA Polymerase In this example DNA polymerase will produce 21 terminated sequences

Cycling Parameters Step 1: 96° C for 1 minute= hot start Step 1: 96° C for 1 minute= hot start Step 2: 96° C for 10 seconds= denaturation Step 2: 96° C for 10 seconds= denaturation Step 3: 50° C for 14 seconds= annealing Step 3: 50° C for 14 seconds= annealing Step 4: 60° C for 4 minutes= primer extension Step 4: 60° C for 4 minutes= primer extension Step 5: Cycle back 24 times to Step 2 Step 5: Cycle back 24 times to Step 2 Step 6: Hold at 4° until purification Step 6: Hold at 4° until purification

Capillary or PAGE Electrophoresis These 21 sequences are separated in a denaturing polyacrylamide electrophoresis. (denaturing, to separate the template). These 21 sequences are separated in a denaturing polyacrylamide electrophoresis. (denaturing, to separate the template). Each sequence will move in the gel as a function of its size, the shortest first. Each sequence will move in the gel as a function of its size, the shortest first. They will pass according to their sizes in front of a excitatory laser beam and a fluorescence detector. As a sequence passes through the excitatory laser beam, it fluoresces according to its terminal ddNTP. The successsion of the fluorescences is recorded. They will pass according to their sizes in front of a excitatory laser beam and a fluorescence detector. As a sequence passes through the excitatory laser beam, it fluoresces according to its terminal ddNTP. The successsion of the fluorescences is recorded.

Instrumentation

Reaction Products are Separated on a Polyacrylamide Gel Each ddNTP is labeled with a flourescent molecule; Cytosine-blue Guanosine-yellow Adenine-Green Thymidine-Red Each different colored bands represents a different sized fragment of DNA, the last nucleotide of which is represented by the dye

Electrophoretogram