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assembling problem in genes


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7 replies to this topic

#1 chicken

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Posted 25 March 2009 - 09:40 PM

hi

I want to know how two or more genes can be assemble without any nucleotide alteration.

Like

If there is gene A . which is divided in 3 parts and present in 3 vectors (a, b, c.) separately.if I want to join what is safest thing I shuld do.

In my mind I think that can be joined by overlapping primers. Forward primer from a n reverse primer contain sequence of a and partially from b.. bt prblm is it may add AT overhangs with taq polymerase.

2nd ly is there any method of assembling , in which pcr run with forward and reverse primer of genes a and b respectively .dose it help in joinig two genes.

Waiting 4 comments and guidenc.

#2 T C

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Posted 25 March 2009 - 09:49 PM

Hey,

If you don't want to alter the nucleotides thinking that it will change the translation and code for different amino acids, just find translationally silent sites, engineer them and proceed.

Best,
TC

#3 chicken

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Posted 26 March 2009 - 08:51 PM

Hey,

If you don't want to alter the nucleotides thinking that it will change the translation and code for different amino acids, just find translationally silent sites, engineer them and proceed.

Best,
TC






in hepatitis c ,i think structural proteins of hepatitis c dont contain silent sites. so i want method that dnt intrupt the sequence.

can i?

Edited by chicken, 26 March 2009 - 11:13 PM.


#4 T C

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Posted 26 March 2009 - 10:47 PM

Hey,

Make silent mutations at the nucleotide level exploiting degeneracy of codons. There are softwares that predict the silent sites for you (seqweb for instance). Just submit the nucleotide sequence and run the program.

Best,
TC

#5 chicken

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Posted 26 March 2009 - 11:47 PM

Hey,

If you don't want to alter the nucleotides thinking that it will change the translation and code for different amino acids, just find translationally silent sites, engineer them and proceed.

Best,
TC






in hepatitis c ,i think structural proteins of hepatitis c dont contain silent sites. so i want method that dnt intrupt the sequence.

can i?



i want to join.exactly.

#6 T C

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Posted 27 March 2009 - 02:34 AM

I didn't get you?

i want to join.exactly.



#7 chicken

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Posted 27 March 2009 - 02:50 AM

i mean i want to assemble 3 genes one after the other without any delition.or any changing.

#8 T C

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Posted 27 March 2009 - 05:55 AM

2 ways to go about it:

1. If u r lucky :)

Find site in the C terminus of 1st gene or Nter of 2nd and Cter of 2nd or Nter of 3rd. Include these sites in the overhang when doing a PCR and use them to stitch. This is what I look for as the first thing.

2. If u r not lucky...which I am always :P,

You have to find translationally silent sites in the C terminus of 1st gene or Nter of 2nd and Cter of 2nd or Nter of 3rd and engineer them in such a way that the reading frame or any amino acid doesn't change. This is what I was referring to till now in all my posts. This is easy to do. Just google how to find translationally silent sites and you will get some software which will do it for you. I use seqweb but that is licensed. I think NEB DNA cutter also finds it.
Do the PCR with these sites and again stitch.

If you have any problems, send me the sequences and i will send you the potentially translational silent sites back.

Best,
TC

i mean i want to assemble 3 genes one after the other without any delition.or any changing.






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