username1058686
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#1
Report Thread starter 8 years ago
#1
hey,

got a few questions about sequencing if anyone could help

1. if you wanna sequence the insulin gene for example, how do you know where to find it on a chromosome to cut it out?

2. if you then wanna sequence it (i.e chain-termination method) you add a primer firstly to the fragment. how do they know what primer sequence to use if they don't know the sequence?

3. when determining the base sequence on a computer, they read the sequence of primers. so are primers fluorescently marked?

thanks in advance(:
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absta101
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Report 8 years ago
#2
1. You use a DNA probe with a label attached so that it can detected. The probes are short strands of DNA that are complementary to the base sequence of a part of the gene you're looking for.

2. Some genes are too long to be sequenced in one go, so you cut them into smaller sections using restriction enzymes (restriction mapping). I'm guessing you should know the start sequence of the fragment you are studying because you know which restriction enzyme you used to make the fragment. Restriction enzymes only cut at specific palindromic sequences so if you know the enzyme used, you should know the palindromic sequence.

To turn the double strand fragment into a single strand fragment you need to break the hydrogen bonds between the nucleotides. You can do this by heating the DNA to around 95C (Celsius).
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