Example DNA
Manipulation - A Summary of Restriction Analysis
Restriction analysis involves restricting /
cleaving DNA into various fragment lengths and
characterizing the fragments through
electrophoresis. The restriction patterns
generated can range from relatively simple to
laughably complex. Here are a few of the issues:
- Number of Fragments - a
restriction endonuclease (RE) which
recognizes a sequence of (8) base pairs in a
human chromosome will produce 6000
fragments. That's an enormous number of
bands to keep track of.
- Electrophoresis - is used to
separate fragments (i.e. DNA is placed in a
agarose gel and fragments are pulled towards
the cathode). Smaller fragments travel
further and are thus separated by size.
- Visualization - after
electrophoresis, fragments are stained with
EtBr (ethidium bromide) and visualized with
u.v. light.
- Figure it out - can be tricky -
by using a wide range of RE (in
specific combinations) one can determine the
identity of base pairs in the sample.
Practical Challenges
of Restriction Mapping
Concept - simple enough: chop up DNA
at specific recognition sites. How? RE cleave at
specific restriction / recognition sites are
used as "landmarks" to infer information about a
DNA molecule.
- Account for all cuts: (i.e.
one cut on linear DNA makes two fragments,
two cuts on linear DNA produces three
fragments. One cut on circular DNA produces
one fragment while two cuts produce two
fragments).
- Account for all base pairs: be aware
that "hidden fragments" can occur by pieces
of the same size from different sections of
DNA. Can appear as a single band, but
contains multiple pieces.
- Try to locate termini - determining the
end fragments (termini) is essential as it
provides a solid reference point.
- Fragments within fragments - try to
determine which fragments appear as
sub-fragments of other restrictions. This
can become very complex!
Simplified
Example Restriction
Let's examine the positions of a theoretical
restriction using ECO R1 on a 10kbp fragment
- We have isolated many copies of the
molecule.
- Sample is treated with Alkaline
Phosphatase (AP) to remove a phosphate group
from the 5' ends.
- Sample is treated with DNA Kinase to add
a radioactive 32P to the 5' ends.
This means all radioactive fragements will
have come from the termini.
- Samples are now treated with one, two,
or three of our RE to generate specific
fragments.
- Electrophoresis - place each sample into
a separate lane on the gel, run the gel and
observe the results of the band migration.
- Interpret results.
http://media.wiley.com/CurrentProtocols/HG/hg0207/hg0207-fig-0001-1-full.gif
Classical Methods of
DNA Sequencing
These techniques are used to establish base
sequences of short (100-5000 bp) fragments.
There are two standard methods:
1. Gilbert-Maxam Method -
modifies specific bases on DNA fragment then
cleaves the sugar-phosphate backbone at the
modified site.
2. Sanger Dideoxy Method -
inhibits replication of DNA fragments at a
specific base.
General Requirements:
- Both methods require homogeneous
single-stranded DNA (heat to melt / separate
strands)
- Strands are separated by electrophoresis
and one band of DNA is removed to provide a
large pool of homologous single-stranded DNA
- Both methods require labeling labeling.
This done through 5'-end labeling by
treatment with alkaline phosphatase (in the
presence of AT32P) to add a
32P to the 5'-end.
Gilbert-Maxam Method
of DNA Sequencing
Treat each of (4) replicate samples with one
of (3) different chemical agents to modify
specific bases. Treatemtns include:
- Hydrazine - breaks pyrimidine rings (T
and C)
- Hydrazine and NaCl - breaks ring of (C)
- Dimethyl Sulphate - methylate 7-Nitrogen
of (G) or 3-Nitrogen of (A)
Next treat these "modified" DNA samples to
cleave the DNA at the modified sites. Low
concentrations are used to modify only a
single base perm DNA molecule. These
secondary treatments include:
- Piperidinee - removes / open (T/C)
rings; break sugar phosphate at this site
- Heat at pH 7 - removes methylated (G)
- Dilute acid wash - remove methylated
purines (A / G), then DNA cleaved by heat /
high pH
Finally conduct electrophoresis. Each band in
the gel represents a "sub-population" of the
treated DNA. Smallest fragments migrate furthest
and the DNA is visualized by radioactivity.
Examine the gel to determine base identities and
sequence. Gel is read from bottom to top
(small to largest fragments) and two lanes
are read to determine base identity (smallest
fragments come from the 5' end).
http://biology200.gsu.edu/houghton/4564%20%2704/figures/lecture%203/Maxam.gif
Sanger Dideoxy
Method of DNA Sequencing
- uses modified nucleotides to block
replication of the DNA molecule and create
characteristic fragments. The method employs
original single-stranded DNA as a template.
- replication of the new strand
(complementary to the template) is
interrupted at specific bases.
- DNA Polymerase replicates by adding
bases 5'--> 3' to the template. A short
primer starts the synthesis.
The method employs a ddNTP (dideoxynucleoside
triphosphate) to stop strand synthesis at
specific points. (i.e. a ddNTP lacks the 3'-OH
group needed to make phosphodiester bonds.
http://www.nwfsc.noaa.gov/publications/techmemos/tm17/figures/moranfig4.htm
Remember - the actual sequence is
complementary to that on the gel!
DNA Libraries
- A "DNA Library" is a collection of DNA
fragments assembled from the genome of a
specific organism.
- The DNA is cleaved into 1000's of
fragments and each is cloned.
- Under optimal conditions, most of
the genome is represented by the "library"
- If the library is constructed from
mRNA it will represent only genes which
are expressed (since mRNA is only
produced for polypeptide synthesis) the
flow:
mRNA -->
mRNA-DNA hybrid --> cDNA (complementary DNA)
http://www.bio.miami.edu/~cmallery/150/gene/sf16x5.jpg
