Biology | Human Molecular Biology Laboratory
L324 | 0589-0590 | Surzycki, S


Course format: Two afternoon laboratories per week (1:00P-5:00P, MW OR
TR, MY 059).

Requirements:  Course in molecular biology or consent of instructor.

Course description: The recent completion of sequencing of human
genome is most important development in history of biological sciences
that would revolutionize not only the understanding of the human
genome, but will change our understanding of life in general.  This
success is based on the development of a number of new molecular
biology techniques that first were applied to the task of studying
human genes but eventually became widely used in all areas of biology.
This course will introduce you to these new techniques.

The human genome is of great interest and is the subject of intensive
basic and applied research.  Molecular biology techniques used in this
research are highly advanced and unique.  Learning these techniques
will permit you to learn basic principles of molecular biology and
also will prepare you to work with the human genome.  These skills are
in great demand by biotechnology, forensic analysis and pharmaceutical
companies.  Besides providing basic experience and understanding of
the principles and practice of cutting edge molecular biology
techniques, the course will provide you with an opportunity to analyze
and study your own genes.  To date no other university offers a course
like this to undergraduates.

Experiments and topics of the course:

1. Preparation of human genomic DNA. Each student will prepare DNA
from his or her own cells.

2. DNA fingerprinting using RFLP analysis with human 33.1 DNA probe.
Techniques used in the course of this experiment:
  Restriction enzyme digestion
  Reverse field agarose gel electrophoresis
  Southern blot transfer
  Preparation of non radioactive probes
  Hybridization and chemiluminescent autoradiography

3. DNA typing by PCR i.e., DNA fingerprinting using single locus
analysis. In this experiment students will type their DNA with probes
used in standard forensic analysis.

4. Linkage disequilibrium analysis using physical markers on
chromosome 12. Loci used for this analysis are Alu CD4 locus and the
TTTTC repeat element. This analysis is based on the paper of Tishoff
et al. in Science: 272;1380-1387 that established a common and recent
African origin for all non-African human populations. Students will
perform PCR analysis of both loci and analyze results by
high-resolution agarose gel electrophoresis. Computer analysis of the
data will consist of calculation of linkage disequilibrium of the
class sample. Results will be compared to the disequilibrium found in
different world populations.

5. Sequencing of human DNA using ABI capillary sequencer and BigDye
technology. Students will prepare their DNA for sequencing using
random sequencing strategy used by Human Genome Project.	Techniques
used in the course of this experiment are:	
  Preparation of a random sequencing library by nebulization
  Preparation of DNA fragments for cloning (DNA end repair)
  Cloning DNA fragments into a sequencing plasmid
  Transformation of E. coli cells by electroporation
  Selection of transformants with inserted DNA
  Preparation of plasmid DNA
  PCR cycle sequencing and preparation of the samples for automatic
sequencing using an ABI capillary sequencer

6. Analysis of sequencing data with computers. Students will carry out
the following analyzes:
  Local alignment analysis with Blast and Fasta
  Determination of expression of sequenced DNA using databases such
as ESTSDB, ALUDB and ICRDB (genes expressed in cancer cells)
  Global alignment analysis e.g., Dot-matrix analysis, GpC island
analysis, ORF analysis
  Analysis of direct and inverted repeats (self-Dot matrix analysis)

7. Determination of human telomere length. The telomere length is a
reflection of the "mitotic clock" of normal somatic cells and
therefore age-dependent. In the course of this experiment, students
will determine telomere length using their DNA. Techniques used are:
  Multi enzyme digestion of genomic DNA
  High-speed agarose gel electrophoresis of DNA fragments
  Turbo-blot transfer and hybridization
  Preparation of DIG-labeled probes by PCR
  Computer analysis and determination of average telomere length

8.	Analysis of actin gene expression. Students will isolate RNA from
their cheek cells and determine actin gene expression using RT-PCR.
Techniques used during this experiment are:
	  RNA isolation and purification
	  Determination of RNA purity and concentration
	  Isolation of polyA+ RNA.
	  RT-PCR reaction and data analysis using high-resolution agarose
gel electrophoresis

Required text:  "Human Molecular Biology laboratory manual" by Stefan
Surzycki. Blackwell Publishing Co., 2003.

Weekly assignments: One chapter from the text and viewing one VCR
"tape of the week."

Exams/papers: Two take home exams and three laboratory reports.