Tuesday, 12 January 2010

GGS LIVE - Southern Blotting

Welcome again in the Biochemistry Methods Section,

Let me introduce today:

Method: Cold Southern Blot

About: Simply sayin Southern Blot is a method were a presence of specific DNA sequence (or its lack) can be detected in the DNA sample of interest. Using the Southern Blot technique we can detect abnormalities/mutations in the whole genomes (karyotypes) like deletions or insertions and many more.

What: Detection of a specific DNA sequence (gene) in the samples form three different cell lines.

How: By cold southern blot technique where non-radioactive DNA probe is used. Major steps of the protocol include: DNA digestion, agarose gel electrophoresis, hybridization with a specifically designed DNA probe and target DNA sequence detection.

Ok so lets start:) Before we go please have a look at these two cool animations about the southern blotting
Southern Blot and Southern Blot 2.

In our case study we will look for presence of a specific gene sequence in three specimens. Each specimen is a different cell line and we will isolate and screen their DNA to look for presence of exemplary gene A.

First thing to do before we start our analysis is to design a probe that will identify DNA sequence of interest. Our probe is of course a short DNA sequence that is complementary to the target sequence. To design the probe we need to know the sequence of the gene A. In this case best probe to use, is a DNA sequence that is a part of our gene A (look at the picture below).

After we design the probe on the computer/paper we have to synthetize it. We obtain the probe by PCR reaction (polymerase chain reaction). To detect the probe we have to label it with a moiety or chemical group that later will be used for detection. We can either use a radioactive or non-radioactive labelling approach for that purpose. In former one, nucleotides bearing a radioactive isotope of phosphorus are used. In the latter, nucleotides bearing unique chemical group/moiety are utilized, which later during the detection step are recognized by specific antibodies

At this stage we have our probe ready and now it is time to prepare DNA samples. We isolate DNA from our specimens and in the next step we have to digest it. We need to digest our DNA in order to release the sequence of interest. Imagine, if we do not digest DNA it will be very hard (almost impossible) to work with such bulk DNA molecule. Additionally, digestion can give us information how big is the DNA fragment that bears our sequence of interest (of course that fragment will be different for various restriction enzymes).
In our study case we will use a BamHI enzyme to fragmentarize our DNA sample. After DNA digestion we separate the DNA fragments by agarose gel electrophoresis, see the picture below.

The "smeary like thing" in lanes Sample A-C, is our digested DNA. Genomic DNA digest result in releasing DNA fragments of  various sizes (ranging from very small ones to big). Remember that genomic DNA is a huge molecule that posseses many BamHI sites.
In the next step we need to transfer the DNA from the gel to the membrane. Membrane is easier to manipulate, because it is less fragile that gel (southern blot membranes are positively charged to attract negatively charged DNA). Just before the transfer gel has to be prepared. Usually it is incubated with different bufferes which remove traces of proteins (residual proteins could interfere with subsequential steps), denaturate and nick DNA. All of that facilitate transfer and further detection of target DNA. There are different ways of the DNA transfer and one of them is the capillary transfer, see the picture below.

After the transfer is finished, DNA is crosslinked to the membrane with UV light (during the crosslink a chemical bond is formed between DNA and a membrane).
Crosslinked membrane is incubated overnight with the probe solution, at a specific temperature which depends on the probe sequence. Hybridizaion is usually performed in hybridizator where membrane is placed in a special tube (see the picture below).

Alternatively membrane can be sealed in the plastic bag containing a probe solution.
After the overnight hybridization mambrane is washed several times to remove unbound/excess probe. In the Cold Southern Blot, probe is detected using specific antibody. Because of that, we need to block the membrane before incubating it with the antibody solution. Blocking prevents antibody to stick/bind to unspecific sites on the membrane. When the membrane is blocked, we can add the antibody solution. Silimarly to western blot a complex between the DNA, probe and antibody is formed during that step (see the picture below and the link to Western Blot technique). Again membrane is washed several times, but this time excess of the antibody is removed.

Antibody used in Southern blot is conjugated to enzyme that catalyze a reaction that emits photon. When the "sandwich complex" is formed, substrate for antibody conjugated enzyme is then applied to the membrane.
Membrane is then sealed in a plastic bag and placed in the cassette. Next, photographic film is applied and cassette is closed and left for several hours. After that time film is developed (see the picture below).

Above you can see a result of our experiment. Our Southern Blot shows that cell lines A and C but not B contains the gene A (target sequence). Additionally we can see that fragment size that bears the target sequence is identicall in specimens A, C and its size can be estimated around 1.8kb (remember that this fragment is specific for BamHI digest and it will be different for other restriction enzyme).
As you see the Southern blot technique is able to detect presence of exemplary gene A.
In the next post about the Gene Targeting I will expose how we can use the Southern blot method in other way.

I hope you enjoy it:)

CyA Soon



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