SGN6237 - Techniques in Molecular Biology and Applications to Gene Expression

 

Course organizers: Andre S. Ribeiro, Olli Yli-Harja

Teacher: Meenakshisundaram Kandhavelu

Teaching assistants: Barathi Muthukrishnan, Kati Waltering, Shanmugapriya Kalaichelvan

Objectives
From this course the student will be introduced to a variety of current techniques in molecular biology. The focus of this course will be manipulation and analysis of gene expression in prokaryotic systems, though eukaryotic tools will be briefly described. Students will become familiar with common wet-lab methods used in various fields in biology, and gain an understanding of the objectives, applicability and limitations underlying each of these methods.

Credits: 3 cr.

 

Lessons content:

 

1st week (1 lesson, 90 min): Genetic manipulation in bacteria I

This lecture will introduce the structure, design and applicability of the bacterial plasmid vector to clone a gene of interest and express protein. Methods used in subcloning, such as PCR, DNA synthesis and sequencing and the process of DNA transfer will be reviewed.

 

2nd week (1 lesson, 90 min): Genetic manipulation in bacteria II

This lecture will continue with techniques introduced in the previous week, through the introduction of genomic insertion by homologous recombination. Methods used to evaluate proper gene insertion, such as Northern and Southern blots, and introduction of mutations by site directed mutagenesis, will be reviewed.

 

3rd week (1 lesson, 90 min): Measurement of mean RNA levels at the Population level.

This lecture will introduce studies of bacterial gene expression. A number of methods used to detect and quantify changing RNA levels due to external stress will be reviewed. Students will be given a summary of multi-cell techniques, including real-time PCR, serial analysis of gene expression (SAGE), and microarray analysis.

 

4th week (1 lesson, 90 min): Measurement of RNA levels at the Single-cell level

This lecture will introduce studies of variability in bacterial gene expression at the single cell level, with an emphasis on cell-to-cell variation. Techniques which are capable of counting individual mRNAs will be evaluated, including fluorescence in situ hybridization (FISH), real-time measurements and single-cell RT-PCR will be reviewed.

 

5th week (1 lesson, 90 min): Cell culturing and staining techniques

In this lecture the students will be introduced to bacterial cell culturing and will culture their own cells, which will be imaged in the next lecture. Also, the students will be introduced to staining techniques and apply it.

 

6th week (1 lesson, 90 min): Microscopy

First, the students will be given instructions on how to operate the microscope. Next, the students will use bright field microscopy to visualize bacteria.

 

7th week (1 lesson, 90 min): PCR techniques

In this lecture the students will perform a PCR measurement.

 

           

Requirements to complete course:

 

Assignments (30% of the final grade), lab report (30% of the final grade) and final project (40% of the final grade). To pass the course, the student is required to execute all three requirements.

Evaluation criteria for the course: Grading from 0 to 5 (0 fails, 1 to 5 passes).

Suggested reading

Type

Reference

Exam material

Language

Article

Raj, A and van Oudenaarden, A., Single-molecule approaches to stochastic gene expression, Annu. Rev. Biophys., 38, 2009, 255-70.

Yes 

English 

Article

Waugh, D. S., Making the most of affinity tags, TIBS, 23(6), 2005, 316-20.

Yes

English 

Article

Fermino, A. M., Fay, F. S., Fogarty, K. and Singer, R. H., Visualization of single RNA transcripts in situ, Science, 280, 1998, 585-9.

No

English 

Article

Reue, K., mRNA quantitation techniques: considerations for experimental design and application, J. Nutr. 128, 1998, 2038-44.

Yes 

English 

Article

Muyrers, J. P. P., Zhang, Youming, and Stewart, A. F., Techniques: recombinogenic engineering – new options for cloning and manipulating DNA, TIBS, 26(4), 2001, 325-31.

No 

English 

Article

Dallas, P. B., Gottardo, N. G., Firth, M. J., Beesley, A. H., Hoffmann, K., Terry, P. A., Freitas, J. R. and Boag, J. M., Gene expression levels assessed by oligonucleotide microarray analysis and quantitative real-time RT-PCR – how well do they correlate?, BMC Genomics, 6(1), 2005, 59-68.

No

English

Prerequisites: Basic knowledge of biology/systems biology. Introduction to Computational Systems Biology (SGN-6056) or equivalent.

Remarks: The course is suitable for postgraduate studies.

Scaling

Methods of instruction

Hours

Lectures

8

Exercises

6

 

Total

14

 

 

Additional information related to course: The course is lectured every year.

 

Course webpage: contains the latest info and some available material for download.

http://www.cs.tut.fi/~sanchesr/SGN-6266/index.htm