DNA sequencing using gel electrophoresis

[mariyam95]

Hi
i need help in this topic plz if u can post any notes and links that might help me coz im seriously desperate now
thanks in advance!

 

[KurayamiKimmi]

Hi
i need help in this topic plz if u can post any notes and links that might help me coz im seriously desperate now
thanks in advance!

is this for bio or chem?

 

[Haider2011]

this may help u in all application topics

 

[mariyam95]

is this for bio or chem?

bio

 

[KurayamiKimmi]

bio

okies here it is

Electrophoresis:
It is a technique that is used to separate DNA fragments of different sizes .
use:
It is used for genetic profiling and for investigating the sequence of bases in a particular length of DNA

Preparing the DNA sample:

• A region of DNA that varies from person to person is chosen , this is known as variable number tandem repeats -VNTR. Only identical twins have the completely same VNTRs.
• The DNA is extracted from the person/crime scene,etc and then the quantity of DNA is increased by the 'polymerase chain reaction' which makes many copies of the DNA therefore even if you start with only a single cell you can have enough sample for electrophoresis.
• The DNA is chopped into pieces using restriction enzyme that are known to cut the DNA close to VNTR sections.

Process of electrophoresis:

• A tank is filled with agarose gel which is very pure, and a direct current is passed continuously through the gel.
• The DNA fragments are all negatively charged due to how they were cut, and so are attracted towards the anode (+ve electrode) and so travel to it , the smaller ones are faster .
• When the current is switched off, the gel contains DNA fragments that have ended up in different places , but this is not visible straight away.

Analyzing the fragments:

• To make them visible, they are first transferred very carefully onto absorbent paper , which is placed on the gel.
• The paper is then heated just enough to make the DNA's two strands separate from each other. Short sequences of single stranded DNA called probes are added, which contain a radioactive isotope. These pair up with the separated DNA strands on the paper , as they have complementary base sequences to VNTR regions.

The paper if now placed on an X Ray film , the radiation emitted by the probes makes the film go dark. So we end up with a pattern of dark stripes on the film, matching the positions that the DNA fragments reached on the gel.

(Do keep in mind that although I've written it in point form , don't do so in the exam...)
There's also a process called 'Southern blotting' do let me know if you'd want that.

 

[fathomspace]

this may help u in all application topics

Thanks a lot,mate..

 

[mariyam95]

okies here it is

Electrophoresis:
It is a technique that is used to separate DNA fragments of different sizes .
use:
It is used for genetic profiling and for investigating the sequence of bases in a particular length of DNA

Preparing the DNA sample:

• A region of DNA that varies from person to person is chosen , this is known as variable number tandem repeats -VNTR. Only identical twins have the completely same VNTRs.
• The DNA is extracted from the person/crime scene,etc and then the quantity of DNA is increased by the 'polymerase chain reaction' which makes many copies of the DNA therefore even if you start with only a single cell you can have enough sample for electrophoresis.
• The DNA is chopped into pieces using restriction enzyme that are known to cut the DNA close to VNTR sections.

Process of electrophoresis:

• A tank is filled with agarose gel which is very pure, and a direct current is passed continuously through the gel.
• The DNA fragments are all negatively charged due to how they were cut, and so are attracted towards the anode (+ve electrode) and so travel to it , the smaller ones are faster .
• When the current is switched off, the gel contains DNA fragments that have ended up in different places , but this is not visible straight away.

Analyzing the fragments:

• To make them visible, they are first transferred very carefully onto absorbent paper , which is placed on the gel.
• The paper is then heated just enough to make the DNA's two strands separate from each other. Short sequences of single stranded DNA called probes are added, which contain a radioactive isotope. These pair up with the separated DNA strands on the paper , as they have complementary base sequences to VNTR regions.

The paper if now placed on an X Ray film , the radiation emitted by the probes makes the film go dark. So we end up with a pattern of dark stripes on the film, matching the positions that the DNA fragments reached on the gel.


(Do keep in mind that although I've written it in point form , don't do so in the exam...)
There's also a process called 'Southern blotting' do let me know if you'd want that.

thank you ...but plz can u help me with DNA sequencing ?

 

[mariyam95]

Thanks a lot,mate..

you welcome

 

[KurayamiKimmi]

thank you ...but plz can u help me with DNA sequencing ?

what about DNA sequencing ?

 

[mariyam95]

what about DNA sequencing ?

i dont understand it -_- i need some notes that explains the process