Broad and Long Term Objective 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 • To Sequence the E. huxleyi cDNA insert in the pMAB plasmid • using cycle sequencing • To learn how to interpret cycle sequencing data • 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 • DNA synthesis reaction whereby fluorescent dideoxynucleotides are incorportated into the newly replicated DNA by DNA polymerase in a primer extension reaction • Thermal cycling reaction
Primer Extension ReactionWhenever a fluorescently labeled dideoxynucleotide is incorporated chain termination occurs DNA Sequencing: Dideoxy Chain Termination Method
DNA Polymerase • an enzyme used in sequencing to extends the 3' end of a primer along a ssDNA template using dNTPs and ddNTPs • DNA pol-1 cannot distinguish a between normal dideoxynucleotides (ddNTPs) and a chemically modified fluorescent ddNTPs • 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 • Primer • Fluorescently labeled ddNTPs • dNTPs • Buffer • 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 2: 96° C for 10 seconds= denaturation • Step 3: 50° C for 14 seconds= annealing • Step 4: 60° C for 4 minutes= primer extension • Step 5: Cycle back 24 times to Step 2 • 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). • 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.
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