Cycle SequencingCycle Sequencing

Broad and Long Term ObjectiveBroad and Long Term Objective

To characterize a single clone from an To characterize a single clone from an Emiliania Emiliania huxleyihuxleyi cDNA library using sequence analysis cDNA library using sequence analysis

Research PlanResearch Plan

Preparation of Competent Cells and Bacterial Transformation

Growth of Transformant and Plasmid MiniPrep

Cycle Sequencing

Sequence analysis

Today’s Laboratory Objectives Today’s Laboratory Objectives

pMAB58

7577 bps

1000

2000

3000

4000

5000

6000

7000

SwaIPpuMI

AhdI

AlwNI

Asp718IKpnIApaIBsp120I

StyIBsmI

PacIBsrGI

BsaBINspV

RsrIIEco47III

BseRISexAIMluI

EcoRIXbaI

BsmIBsrGISmaI

BsaBIEcoRI

NotIMluI

BsrGIAatII++

SacII

BamHI

DraIII

Bsu36IXbaI

BspEISnaBI

ARS4/Cen6

Amp

ori

pADHNLS

ADattB1

ccdB

attB2

Ter ADH

pT7

F1

TRP1

1.1. To Sequence the E. huxleyi cDNA insert in the pMAB plasmid To Sequence the E. huxleyi cDNA insert in the pMAB plasmid

using cycle sequencingusing cycle sequencing

2.2. To learn how to interpret cycle sequencing dataTo learn how to interpret cycle sequencing data

3.3. To learn how to characterize a DNA sequence using various To learn how to characterize a DNA sequence using various web-based bioinformatics toolsweb-based bioinformatics tools

What is Cycle Sequencing?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 DNA synthesis reaction whereby fluorescent dideoxynucleotides are

incorportated into the newly replicated DNA by DNA polymerase in a incorportated into the newly replicated DNA by DNA polymerase in a primer extension reactionprimer extension reaction

Thermal cycling reactionThermal cycling reaction

Primer Extension ReactionPrimer Extension Reaction

Whenever a fluorescently labeled dideoxynucleotide is incorporated chain Whenever a fluorescently labeled dideoxynucleotide is incorporated chain termination occurstermination occurs

DNA Sequencing: Dideoxy Chain Termination Method

DNA PolymeraseDNA 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 ComponentsPrimer Extension Reaction Components

DNA templateDNA template PrimerPrimer Fluorescently labeled ddNTPsFluorescently labeled ddNTPs dNTPsdNTPs BufferBuffer Amplitaq= termal stable DNA PolymeraseAmplitaq= termal stable DNA Polymerase

In this example DNA polymerase will produce 21 terminated sequences

Cycling ParametersCycling Parameters

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

Capillary or PAGE ElectrophoresisCapillary or PAGE Electrophoresis

These 21 sequences are separated in a denaturing polyacrylamide electrophoresis. These 21 sequences are separated in a denaturing polyacrylamide electrophoresis. (denaturing, to separate the template).(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 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 fluorescence detector. As a sequence passes through the excitatory laser beam, it fluoresces according to its terminal ddNTP. The successsion of the fluorescences is fluoresces according to its terminal ddNTP. The successsion of the fluorescences is recorded.recorded.

InstrumentationInstrumentation

Reaction Products are Separated on a Reaction Products are Separated on a Polyacrylamide GelPolyacrylamide Gel

Each ddNTP is labeled with a flourescent molecule;

Cytosine-blueGuanosine-yellow Adenine-GreenThymidine-Red

Each different colored bands represents a different sized fragment of DNA, the last nucleotide of which is represented by the dye

ElectrophoretogramElectrophoretogram