RESTRICTION MAPPING
the process of obtaining structural information on a piece of
DNA by the use of restriction enzymes.
A restriction map is a map of known restriction sites within a
sequence of DNA
Restriction mapping steps
1. Breaking DNA into pieces
2. identifying the locations of the breakpoints.
Restriction Enzymes
endonucleases that recognize specific 4 to 8 base regions of DNA. restriction sites.
evolved as a bacterial defense against DNA bacteriophage
Recognizes Palindromic seq,
each strand of the DNA can self-anneal and the DNA forms a small cruciform
structure
Hundreds of restriction enzymes that have been isolated and each one
recognizes its own specific nucleotide sequence.
Sites for each restriction enzyme are distributed randomly throughout a
particular DNA stretch. Digestion of DNA by restriction enzymes is very
reproducible; every time a specific piece of DNA is cut by a specific enzyme,
the same pattern of digestion will occur.
Uses of Restriction Mapping
for many techniques used to manipulate DNA.
One application is to cut a large piece of DNA into smaller fragments to allow it to be
sequenced. Genes and cDNAs can be thousands of kilobases long (megabases -
Mb); however, they can only be sequenced 400 bases at a time. DNA must be
chopped up into smaller pieces and sub cloned to perform the sequencing.
an easy way to compare DNA fragments without having any information of their
nucleotide sequence.
The sum of the individual fragments =size of the original fragment
If not there are two likely problems.
In one case, some of the smaller fragments may have run off the end
of the gel.
1. the gel was not dense enough and therefore was unable to
resolve fragments close in size.
2. This leads to a lack of separation of fragments which were close in
size.
Course Contents
1.
2.
3.
4.
5.
6.
7.
8.
DNA and RNA isolation
Quantification of DNA and RNA
Primer designing
PCR
Electrophoresis
Sequencing
Karyotyping
Restriction Mapping
9. Flow cytometry
10.Hybridization
a. Western blotting
b. Southern blotting
c. Northern blotting
d. FISH
11.Transfection
21.Tissue culturing
12.Transduction
22.Slide Preparation and Cell Stains
13.Transformation
23.Agar plate preparation and streaking for
14.Cloning
the purpose of individual colony isolation
15.Microarrays
24.Bacterial Growth on selective agar
16.Chromatography
25.Quantification: Colony Forming Units
17.Immunochemistry
(CFU)
18.ELISA
26.Dilution Plating
19.Bioinformatics and techniques
27.Identification
20.Ethical issues
colonies
and
characteristics
of
Lab Work:
•DNA extraction
•Quantification
•PCR
•Electrophoresis
•ELISA
•Agar plate preparation
•Streaking
•Transfection
•Cloning