Biotechnology in the NSS biology curriculum Daniel K. L. Lee, Ph. D. Department of Applied Biology and Chemical Technology The Hong Kong Polytechnic University

Rapidly expanding area Understand the principles of the techniques Familiarize oneself with the applications Use the appropriate instruments and the proper techniques PCR Gel electrophoresis

The message is To guide the students through the PCR and gel electrophoresis experiments with the use of appropriate instruments and proper techniques so that the students will understand the principles and the applications of these techniques.

To carry out the experiment, we need The appropriate instruments The necessary reagents and materials A procedure that works The proper techniques

Amplifying DNA using PCR and separating DNA fragments with gel electrophoresis

The instruments Micropipettes PCR machine (thermal cycler) Gel electrophoresis system  Gel tank  Gel tray  Power supply Microcentrifuge

Instrument Approximate cost MicropipettesHK$800 – 2000 Thermal cyclerHK$30,000 upwards Gel tank + trayHK$2,000 upwards Power supplyHK$5,000 upwards MicrocentrifugeHK$2,000 upwards These instruments are available from most biotechnology companies that sell equipments. The price of each instrument varies from brand to brand, and also from model to model. One needs to strike a balance between functions and cost. Usually the basic models are good enough for these experiments in secondary schools.

Reagents and materials Micropipette tips Microtubes Template DNA Primers Taq DNA polymerase Deoxynucleotide triphosphates PCR buffer Agarose Electrophoresis buffer Gel loading dye DNA stain

Reagents and materials Not as expensive as one may think Need to source from different companies Need to test and optimize the conditions Most of the reagents available are mainly for research purposes, not for secondary school teaching. For PCR, you need to decide on  The template DNA [human, mouse, plant DNA?]  The pair of primers [defines the segment of DNA to be amplified.]

A procedure that works Different conditions are required for different DNA samples, different primers, and reagents from different sources. A procedure that works for human DNA with a specific pair of primers may not work with another pair of primers. A procedure and a pair of primers that work for human DNA may not work with mouse DNA. Optimization by test runs required.

Proper techniques Using the micropipettes Using the thermal cycler Mixing the reagents in the microtubes Using the microcentrifuge Preparing the agarose gel Loading the samples into the wells Staining the gel Proper training for teachers and technicians

DNA workshops in PolyU

We have undergraduate students who may work as part-time TAs to help you with the preparation, demonstration, and teaching of the experiments. These are students who have organized the summer DNA camps and workshops, prepared the experiments and taught the students and teachers.

The experiment is done! But Learning has just begun!

On Gel Electrophoresis Which direction did the DNA moved, towards the anode or the cathode? Why is DNA moving in this direction?  What is the charge of DNA molecules?  Which part of the DNA does the charge come from? [The phosphate groups along the sugar-phosphate backbone] One phosphate group per base; longer DNA, more phosphate groups, more charges. Charge/mass ratio is constant for all DNA, irrespective of length. The mobility solely depends on how easy a DNA molecule moves through the pores of the gel; smaller DNA easier; larger DNA more difficult.

On the PCR Results Why is there no band in B and C? [No reagents; no template DNA] Why do we need to include B and C in the experiment? [Controls] Why are there two bands in A?  two different DNA, of different sizes  two different PCR products Why are there two different PCR products?  Two different templates or two different pairs of primers  Diploid genome; Heterozygous “Aa” for this locus; basic genetics concepts.

On the design of the experiment Why do we need to run the gel?  To separate the DNA fragments by size difference  To check if we have the DNA fragment of the correct size  To compare between different samples Why do we need to do PCR?  To amplify the DNA  To do DNA fingerprinting  To detect microbes  To detect GM food What DNA shall we use as template? Which DNA region shall we PCR? What primers shall we use? This is all about applications.

Biotech teaching made easy by packages available from the Life Science Education division of a biotechnology company  Genes in a Bottle Kit [S1, S2]  pGLO Bacterial Transformation Kit [S2, S3]  Restriction Digestion and Analysis of Lambda DNA Kit [S4, S5]  Crime Scene Investigator PCR Basics Kit [S5, S6]

You are always welcome to contact me if you need more information or help!