By hybridization of labeled nucleic acid probe, DNA can be detected by blotting methods (called Southern blots, after the inventor Dr. Southern). After a nucleic acid sample is run on a gel, and a picture is taken to establish the mobilities of known molecular weight standards, the DNA in the gel can be "blotted" onto a special type of membrane (nitrocellulose or nylon-based membranes) so that an imprint of the separated nucleic acids is made.
Then, a labeled nucleic acid (marked with either radioisotope or chemical label) is allowed to anneal or hybridize to the DNA on the filter -- this "probe" bonds only to the DNA to which it is complementary, by base-pairing rules. Once the excess probe is washed away, and the hydrogen bonded probe is detected, an image of the gel can be created that shows where sequences complementary to the probe happened to be when you halted the electrophoresis. The great advantage of this method is that you can detect small quantities (1 to 10 pg per band) of a specific nucleic acid, so it is at least 1000 times more sensitive than ethidium bromide.
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There are two common ways of transferring nucleic
acids from a gel to a membrane.
- One is to use an electrophoresis tank to apply an electric field across the face of the gel, causing the molecules to leave the gel (in the direction of the membrane).
From: The MIT hypertextbook
Another is to use the capillary action of a solution to wash the nucleic acids through the gel and onto the membrane. We do this by placing the gel on a paper wick, over a reservoir of liquid, and placing the nitrocellulose or nylon membrane on top of the gel, then placing paper towels on the membrane so that it soaks up the liquid (that's why we call it a "blot"). As the liquid is sucked through the membrane, more is drawn up into the gel through the paper wick. The net result is a rush of liquid passing through the gel and membrane, washing the DNA onto the membrane where it will stick. Some people skip using a reservoir of liquid, and just use the liquid in the gel to transfer the nucleic acids during blotting.
From: The MIT hypertextbook
Preparation of a labeled probe
A probe sequence can be labeled by several methods:
- "Nick translation" in which a double stranded DNA sequence is nicked with a limiting amount of a nonspecific nuclease such as DNase I. These breaks in the phosphodiester backbone are points of entry for the enzyme DNA Polymerase I (holoenzyme, which has 5' to 3' and 3' to 5' exonuclease activities, in addition to a 5' to 3' synthetic activity). The nicks in the DNA are "translated," or moved along the backbone, by incorporation of new nucleotides. If one of the nucleotide substrates (say dCTP) is labeled with radioisotope (for example the alpha phosphate can be P-32), or a chemical group (digoxygenin or biotin), the labeled groups will be incorporated into short stretches of DNA. This process is called "nick translation" to describe the action of the polymerase, but since it doesn't always work that well we used to call it "nicht translation" in grad school!
from: Washington University
- "Random oligo primed synthesis" in which a DNA is denatured into single strands and annealed to random hexamer oligonucleotides. These random primers can then be extended using Klenow enzyme, incorporating labeled nucleotides (as above).
From: The MIT hypertextbook
Single stranded nucleic acids hydrogen bond to each other efficiently, following Watson-Crick base pairing rules, at approximately 20-25 degrees centigrade below their melting point. To say it a different way, when hybridizing a probe to the DNA or RNA on a membrane, we adjust the solution conditions (for example, the salt concentration) so that the melting point of the nucleic acids is approximately 20-25 degrees higher than the incubation temperature. Lowering the salt concentration lowers the melting point, as does the addition of formamide. A typical condition for hybridization is:
-
6x SSC
0.2% SDS
1x Denhardt's blocking solution, or 1% w/v milk
10-50 ng/ml probe (denatured first!)
65 oC incubation, with agitation, for 18-24 hours.
Following a period of hybridization, it is necessary to wash off the probe that is
loosely bound to the membrane (i.e. nonspecifically bound). This is typically done
by washing the filter several times at 65 C in decreasing salt concentrations (i.e.
3xSSC/0.2% SDS, then 1x SSC/0.2% SDS, etc...).
Detection (a couple of examples
only)
- If the probe is radioactively labeled, the membrane can be placed on X-ray film, and an image of the bound probe can be established by autoradiography.
- If the probe is chemically labeled (for example, digoxygenin labeled), the filter is exposed to an anti-digoxygenin antibody conjugated to an enzyme such as alkaline phosphatase. Once the excess antibody is washed away, the substrates NBT and BCIP can be applied, which will be converted to an insoluble blue dye by the alkaline phosphatase. Therefore, the position of the bound probe is indirectly indicated by the enzymatic reaction.
What is happening during hybridization of a probe?
A single stranded probe is finding DNA sequences that it can hydrogen bond to, and
these hydrogen bonds cause the two molecules to stick together. In general, the DNA
on the filter is denatured into single strands before it is blotted, and the probe
is boiled (denatured) before it is added to the hybridization mixture. The probe
is labeled on both strands (usually), so either strand can hybridize with a DNA strand
on the filter. On the other hand, the labeled DNA can also re-anneal to its partner
in solution -- the opposite labeled strand of DNA. What happens then is that the
double stranded pair in solution does not contribute to the signal on the membrane
-- they are essentially lost from the reaction.
Important concepts.
The concentration of the probe in the hybridization reaction is important, because
there has to be some reasonable chance of the probe molecule "bumping into"
the target molecule. If the concentration of probe is low, the time it takes to anneal
a significant number of probe molecules to a target band is increased. That is, you
have to hybridize your probe to your blot for a longer time - perhaps many days instead
of just overnight. If you hope to use the intensity of hybridization to reflect the
amount of material in your target band, the total amount of your probe should be
in excess over the amount of target DNA on your blot. If you do not have excess probe,
the linearity of the hybridization signals will be questionable.
The labeled probe, once bound to a target sequence, will "broadcast" its presence by the fact that it is either radiolabeled or chemically labeled. In either case, the signal strength will depend on how much label was incorporated into the probe when it was synthesized. If only one nucleotide is labeled per probe strand, on the average, your hybridization signal will be exactly 10 times lower than if 10 nucleotides had been labeled per strand. The amount of label incorporated is called the "specific activity" of your probe. When working with radioactive materials your safety is extremely important. There are several excellent non-radioactive approaches towards probing a Southern blot, including chemiluminescence and the BCIP/NBT Genius method we use.
Analysis of Southern blots
Here's a simple example. Suppose the map below represents a plasmid pA1.
You perform a Southern blot, using the probe sequence marked ABC, and get the following result (compare the map printed above with the gel and Southern blot results below)
Note that there are several important concepts here:
- Not every band that is detected by ethidium bromide is detected by the ABC probe.
- There is some variation in intensity of hybridization (particularly in the case of the 4.0 kbp band, which is not heavily labeled by the hybridized probe.
How do you explain these two points?
Examples
Try the following problems at the University of Arizona site: