Biology | Human Molecular Biology Laboratory
L324 | 0659-0660 | Surzycki


Course format: One lecture (50 minute) and two afternoon laboratories per
week (1:30P-5:30P, MW OR TR, JH 131).

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:
o  Restriction enzyme digestion
o  Reverse field agarose gel electrophoresis
o  Southern blot transfer
o  Preparation of non radioactive probes
o  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:
o  Preparation of a random sequencing library by nebulization
o  Preparation of DNA fragments for cloning (DNA end repair)
o  Cloning DNA fragments into a sequencing plasmid
o  Transformation of E. coli cells by electroporation
o  Selection of transformants with inserted DNA
o  Preparation of plasmid DNA
o  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:
o  Local alignment analysis with Blast and Fasta
o  Determination of expression of sequenced DNA using databases such as
ESTSDB, ALUDB and ICRDB (genes expressed in cancer cells)
o  Global alignment analysis e.g., Dot-matrix analysis, GpC island
analysis, ORF analysis
o  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:
o  Multi enzyme digestion of genomic DNA
o  High-speed agarose gel electrophoresis of DNA fragments
o  Turbo-blot transfer and hybridization
o  Preparation of DIG-labeled probes by PCR
o  Computer analysis and determination of average telomere length

8. Single locus polymorphism analysis (SLP). SLP present on the X
chromosome will be analyzed (#618). Students will use their DNA for this
analysis. Data of this analysis will be submitted to SNP population
database at NCBI.

9.	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:
o  RNA isolation and purification
o  Determination of RNA purity and concentration
o  Isolation of polyA+ RNA.
o  RT-PCR reaction and data analysis using high-resolution agarose gel
electrophoresis

Required text:  "Basic Techniques in Molecular Biology" by Stefan Surzycki.
Springer-Verlag. 2000.

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.