Southern Blot
- Created by: 08rmorris
- Created on: 28-03-15 13:44
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- Southern Blot
- Used to detect a specific target sequence in samples of DNA
- DNA samples separated in an electrophoresis gel is fragile, and the separated DNA fragments continue to diffuse through it blurring the bands
- So, in a blot, the DNA is transferred to a stronger substrate, where it can be fixed
- The target sequence is then detected using a DNA probe
- A DNA probe is simply a short length of single-stranded DNA with a label attached
- There are two common types of label used:
- - A radioactively-labelled probe (synthesised using the isotope 32P) can be visualised using autoradiography
- - A flourescently-labelled probe will emit visible light when illuminated with invisible ultraviolet light. Probes can be made to fluoresce with different colours
- If a probe is added to a mixture of different pieces of single-stranded DNA it will anneal to any fragments of DNA containing the complementary sequence
- forms regions of double-standed DNA (hybrid) the process is therefore called hybridisation
- There are two common types of label used:
- A DNA probe is simply a short length of single-stranded DNA with a label attached
- The target sequence is then detected using a DNA probe
- So, in a blot, the DNA is transferred to a stronger substrate, where it can be fixed
- DNA samples separated in an electrophoresis gel is fragile, and the separated DNA fragments continue to diffuse through it blurring the bands
- METHOD
- 1. DNA is extracted from the source cells and amplified by PCR to make enough DNA for the hybridisation
- The DNA samples are then digested by a restriction enzyme into many small fragments, and the fragments separated on an electrophoresis gel
- 2. The gel is placed in an alkali solution, which breaks the H bonds between the DNA bases causing the strands to separate
- 3. A thin sheet or nitrocellulose is placed on top of the gel and covered with a stack of paper towels, weighted down to make good contact.
- The alkali solution is drawn up through the gel to the paper towels by capillary action, bringing the DNA with it.
- The negatively charged DNA sticks to the positively charged nylon membrane
- 4. After a few hours the DNA fragments are all transferred to the nylon sheet
- The nylon sheet is separated from the del and treated with UV light to fix the DNA molecules
- 5. The nylon sheet is placed in a plastic bag containing a solution of labelled probes and mixed thoroughly.
- The probes will anneal by complementary base-pairing to DNA fragments in the nylon membrane that have a complementary sequence
- Forming hybrid DNA molecules stuck to the nylon sheet (but they can't be seen)
- 6. The location of the hybrid DNA can be visualised by different methods, depending on the label used.
- If the probe were radioactive then they can be visualised by autoradiography, and the probes show up as band on photographic film
- If the probes were fluorescent then they can be visualised as bands of light when illuminated by ultraviolet light.
- If the probe were radioactive then they can be visualised by autoradiography, and the probes show up as band on photographic film
- The probes will anneal by complementary base-pairing to DNA fragments in the nylon membrane that have a complementary sequence
- The alkali solution is drawn up through the gel to the paper towels by capillary action, bringing the DNA with it.
- 3. A thin sheet or nitrocellulose is placed on top of the gel and covered with a stack of paper towels, weighted down to make good contact.
- 1. DNA is extracted from the source cells and amplified by PCR to make enough DNA for the hybridisation
- The southern blot has many uses
- - to identify fragments containing a particular gene out of the thousands of restriction fragments formed from an entire genome
- - to identify genes in one species that are similar to those of another species. Genes are remarkably similar in sequence form one species to another
- this has aided the identification of human genes
- - To identify the short DNA sequences used in DNA fingerprinting
- - to screen for genetic diseases
- Used to detect a specific target sequence in samples of DNA
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