서던 블롯: DNA 단편 검출 및 분석

### 서던 블로팅: 서던 블로팅: 종합적인 개요
* "남부 블롯은 **DNA의 정체성, 크기, 풍부도에 대한 정보**를 알려주는 분자생물학의 고전적인 기법입니다."
* ⚙ 이 과정은 특정 인식 부위에서 DNA를 단편으로 절단하는 **제한 효소**를 사용하여 DNA 샘플을 분해하는 것으로 시작됩니다.
* 🔬 다음으로, DNA 단편을 겔 전기영동으로 분리하는데, 이때 **작은 단편은 전하 대 질량비로 인해 더 빠르게 이동**합니다.
* 🌡 ️ 전기영동 후, DNA 조각을 알칼리성 용액으로 변성시켜 **이중 가닥을 분리**합니다.
* 📤 그런 다음 변성된 DNA는 **모세관 작용과 이온 교환 상호작용**을 통해 막으로 옮겨집니다.
* 일단 DNA가 옮겨지면 **자외선을 사용하여 멤브레인에 영구적으로 부착**됩니다.
* 🎯 그런 다음 멤브레인을 방사성 표지된 DNA 프로브와 하이브리드화하여 **특정 DNA 서열을 감지**합니다.
* 📸 마지막으로 자가 방사선 촬영 방법을 사용하여 **막에서 방사능으로 표지된 DNA의 위치**를 시각화합니다.

서던 블로팅 기술은 DNA 분해, 겔 전기영동, 멤브레인으로의 DNA 이동, 프로브와의 혼성화, 유전자 식별을 위한 자동 방사선 조영술로 이루어집니다.

Southern blot is a classic technique in molecular biology that reveals information about DNA identity, size, and abundance, and it can be used for detection of a specific DNA sequence in DNA samples. The first step in Southern blotting is the digestion of the DNA samples with an appropriate restriction enzyme. A restriction enzyme, also called restriction endonuclease, is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Once the restriction enzyme is added, the samples are incubated at 37 degrees celsius overnight. Restriction enzymes recognize a specific sequence of nucleotides and produce a double-stranded cut in the DNA. Therefore, various DNA fragments of different sizes are obtained. Then, the DNA fragments produced by restriction endonuclease digestion are separated by gel electrophoresis. For gel electrophoresis separation, a loading buffer is added to the DNA samples, and it is used as a tracking dye, which migrates in the same direction as DNA.

allowing the user to monitor the progress of the separation. Agarose gel electrophoresis is most commonly used to separate mixtures of DNA fragments of varying sizes. During electrophoresis, a molecular-weight size marker, known as a DNA ladder, is commonly used to determine the size of DNA fragments in the samples. The DNA ladder is added into well at one end of the gel. Once the molecular-weight size marker is added, the DNA samples are loaded into wells. Then, an electric current is applied to pull the samples through the gel. Based on their charge and size, the molecules will travel through the gel at different speeds, allowing them to be separated from one another. The phosphate backbone of the DNA molecule is negatively charged; therefore, when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because all DNA fragments have the same amount of charge per mass, small fragments move through the gel faster than large ones. After the electrophoresis is complete, the molecules in the gel can be stained to make them visible.

when the gel is stained with an intercalating dye, such as Ethidium bromide, the DNA fragments can be seen under UV light as bands, each representing a group of same-sized DNA fragments. After the separation of the DNA molecules, the double-stranded DNA fragments are denatured with an alkaline solution consisting of sodium hydroxide. The gel is soaked in the alkaline solution with gentle agitation. At an alkaline pH, hydroxide ions are predominant; consequently, guanine and thymine will be deprotonated and exist as negatively charged conjugate bases. This process breaks the hydrogen bonds between the oligonucleotides, causing the separation of the two DNA strands. After the denaturation treatment, the alkaline solution is removed. Then, a neutralizing solution is used to neutralize the pH of the gel. This allows for a more efficient transfer of DNA in Southern transfers. While the gel is neutralizing, filter paper and membrane are prepared for Southern blotting. To transfer the denatured DNA to the membrane

a transfer buffer, a solid support, and a sheet of blotting paper act as a wick for the transfer solution. The wick is placed over the solid support in the transfer reservoir so the ends will be in the transfer buffer. Then, it is wetted with the transfer solution. Next, pieces of extra thick blotting paper are placed on top of the wick. Then, they are wetted with the transfer solution. Once the gel is neutralized, it is placed on the thoroughly wetted wicking paper. Then, a sheet of nylon or nitrocellulose membrane with the same size as the gel is pre-wetted with the transfer solution and placed on top of the gel. Next, pre-wetted pieces of extra thick blotting paper are placed on top of the membrane. The exposed areas of the wick are covered with strips of plastic wrap to prevent transfer buffer from bypassing the gel during the transfer process. Finally, a dry stack of paper towels is placed on top of the membrane and gel.

then a glass plate is placed on top of these sheets paper, with a weight to maintain tight contact between the gel and membrane. Buffer transfer by capillary action from a region of high water potential to a region of low water potential is then used to move the DNA from the gel onto the membrane. Consequently, ion exchange interactions bind the DNA to the membrane due to the negative charge of the DNA and positive charge of the membrane. The transfer is allowed to proceed overnight, then once it is completed, the blotting material and membrane are carefully removed from the gel. Next, the membrane is briefly rinsed to remove any agarose that may be stuck during the transfer, then it is exposed to ultraviolet radiation to permanently attach the transferred DNA to the membrane. After attachment of the DNA fragments to the membrane, hybridization with radiolabeled DNA probes is performed. The membrane is placed in a bottle containing a pre-hybridization solution, which is used to reduce non-specific hybridization with probe. Next, the bottle is incubated in hybridization oven.

at 42 degrees Celsius for 2 hours. Once the incubation is complete, the pre-hybridization solution is removed. Then a hybridization buffer is added into the bottle. Next, labeled DNA probes are added to the hybridization solution. The probes are fragments of DNA of variable length, which can be radioactively or fluorescently labeled. Once the hybridization probes are added, the bottle is incubated overnight in the hybridization oven at 42 degrees Celsius. DNA contains a large quantity of phosphorus in the phosphodiester linkages between bases in the oligonucleotide chain. DNA can therefore be tracked by replacing its non-radioactive phosphorus with radioactive phosphorus-32. The radioactively labeled DNA probes hybridize to their complementary sequences in the DNA fragments. After the hybridization of each probe to its target sequence, the hybridization solution is removed. Next, a wash buffer is added into the bottle. Then the membrane is incubated at 52 degrees Celsius for 30 minutes. The washing process is repeated three times to remove unbound and weakly binding probe.

After hybridization, an autoradiography method is carried out to identify the location of radioactively labeled DNA in the membrane. The Southern Blot filter is placed inside a light-proof cassette box. Then, an X-ray film is laid over the top. The cassette is closed and left for several hours to several days. The radioisotope-labeled DNA exposes the film, which when developed, shows a pattern of black bands that indicate the positions of labeled DNA in the blot membrane. Subsequently, this information can be used to determine which gene is present in each sample. Also, the position of each gene can be identified in the gel. Consequently, after electrophoresis, bands of interest can be cut out from the gel so that each gene can be isolated for further analysis.

Southern Blot Method - Animated Video

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Biology with Animations

분자생물학에서의 서던 블롯 과정

남부 블롯은 샘플에서 특정 DNA 서열을 검출하는 데 사용되는 기술로, 제한 효소로 DNA를 분해하는 과정을 포함합니다.

그런 다음 아가로스 겔 전기 영동을 사용하여 소화된 DNA 조각을 분리하여 조각 크기를 결정할 수 있습니다.

전하와 크기에 따라 DNA 조각을 분리하기 위해 겔에 전류를 흘려 작은 조각이 더 빠르게 이동합니다.

그런 다음 전기 영동이 완료된 후 염색을 사용하여 DNA 조각을 볼 수 있게 합니다.

DNA 조각은 인터칼레이팅 염료로 염색하고 젤에서 분리한 후 밴드 형태로 시각화됩니다.

그런 다음 알칼리성 용액을 사용하여 이중 가닥 DNA를 변성시켜 가닥 사이의 수소 결합을 끊습니다.

변성된 DNA는 심지, 전사 버퍼, 블롯팅 페이퍼를 사용하여 멤브레인으로 옮기는 서던 블로팅(Southern blotting) 과정을 거칩니다.

DNA 블 롯팅 프로세스: 단계별 가이드

유리판과 추를 사용하여 젤과 멤브레인을 함께 눌러 DNA를 옮깁니다.

모세관 작용에 의해 DNA가 겔에서 멤브레인으로 이동하여 이온 교환 상호작용이 DNA를 결합합니다.

그런 다음 멤브레인을 자외선에 노출시킨 후 DNA 프로브와 혼성화하고 결합되지 않은 프로브를 제거하기 위해 세척 과정을 거칩니다.

자동 방사선 촬영을 통한 DNA 식별 과정

자동 방사선 촬영은 혼성화 후 멤브레인에서 방사능으로 표지된 DNA의 위치를 찾는 데 사용됩니다.

노출된 X-레이 필름에는 검은색 띠가 표시되어 표지된 DNA의 위치를 나타내며, 이는 각 샘플에서 유전자의 존재와 위치를 파악하는 데 도움이 됩니다.

이 정보를 통해 전기영동 후 추가 분석을 위해 겔에서 유전자를 분리할 수 있습니다.