Can anyone clarify and help me learn

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Biolink
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Can anyone clarify and help me learn

My professor gave me three amino acid sequence and using the codon table he asked me to design a set of DNA oligonucleotide probe which can be used to detect in a library. He also asked me to find out the preferable oligonucleotide probes used to screen a library.

The peptide sequence given by him is
(a)        H-C-W-K-M
5¢- CAU – UGU – UGG – AAA – AUG - 3¢
            OR
5¢- CAC – UGC – UGG – AAG – AUG - 3¢
In this way I also have
(b)       R-S-L-L-S
(c)        D-A-Q-W-Y

 Similarly I got sequence for R-S-L-L-S and those sequences are
5¢- CGU – UCU –CUU—CUU – AGU- 3¢
5¢- CGC – UCC –CUC—CUC– AGC- 3¢
Like this two more combinations are also possible,
Similarly I have 4 possible sequences for D-A-Q-W-Y
Keeping this information how to design a oligonucleotide probe as well how to choose the preferable probe which can be used to screen a library. Kindly explain me from the basics so then I can understand the concept well. Please help me.
 

Chuck Miller
Chuck Miller's picture
 You are soooo close.....

 You are soooo close.....
You listed the RNA codons.....now change these to DNA.
Different organisms use different nucleotides with different frequencies for encoding a particular amino acid.
Try looking up something called a codon usage table for your particular organism / virus of interest. 
CM

Biolink
Biolink's picture
Thank you for your reply,

Thank you for your reply, well as you say ill convert them for DNA, i have to replace the uracil with thiamine for all the pairings and after getting that ill be having hand full of probes, you could see three peptides represnting one particular protein,for each of the three peptides mentioned in the query ill design a set of DNA oligonucleotide probes that can be used to detect the gene in a library. Which of the three sets of oligonucleotide probes would be preferable to screen a library? Explain.

Chuck Miller
Chuck Miller's picture
 The answer will depend on

 The answer will depend on the purpose of your library --are you looking for something abundant/rare/ a particular gene? --and on codon useage in the organism of interest. If you know the gene that you are trying to probe then it is easy--the sequence you pick will be the one that matches the gene of interest. If the gene is known in one species but not another then you can guess--based on codon usage in the particular organism -- which might be best. Read and understand codon useage if you don't know about it. 

The FFM
The FFM's picture
When designing oligos like

When designing oligos like this you need to consider 3 things:
1) "codon usage" in the species from which the library is derived
2) intercodon dinucleotide frequency
3) potential probe self-complementarity
I'll let you do some background research of your own on those terms.
 
In terms of the possible number of oligo designs for each sequence just consider your first sequence HCWKM
There are more than two possibilities. Three of the five codons have two possible codons.  You have a reverse translation product CAYTGYTGGAARATG where Y= T/C and R = A/G so you have 23 possibilities for your oligo sequences
 
 

Chuck Miller
Chuck Miller's picture
 He asked which SET of probes

 He asked which SET of probes would you screen--correct?
Which set has the fewest possibilities (least work/is easiest) to screen?
 

Biolink
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Thanks a lot now ill search

Thanks a lot now ill search as FFM said and i hope i can justify my answer after reading that and thanks a lot miller for your clue.

The FFM
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here is an online codon usage

here is an online codon usage database http://www.kazusa.or.jp/codon/

Biolink
Biolink's picture
Well can you also give me a

Well can you also give me a clue for another question where my professor provided me two fungal speacies names and he siad that it lives in extreme hot condition around 95 degree celsius. He gave the property of the two fungal s[ecies as shown below,

Property

F. cattoriae

W. gravinius

 

 

 

Genome size

1 MB

3 MB

Repetitive DNA?

20% of genome contains
large stretches of CG repeats

< 0.1% of genome

By sequencing and examining their genomes, he said that I can understand why these species can live in extreme environments. For that he said that he will give me chance to sequence only one genome, I don’t how should I choose the best one and would like your input as to which species should be sequenced and can I apply shotgun strategy whether it will work in this case.

Biolink
Biolink's picture
Thank you FFM for your Codon

Thank you FFM for your Codon usage database

The FFM
The FFM's picture
Fact: sequencing through

Fact: sequencing through repetative and/or GC rich regions sucks

Biolink
Biolink's picture
Hmmmmmmm like the previoua

Hmmmmmmm like the previoua question can you tell me what information i should read to understand and answer this question, bcoz you say that repetetive or CG regions will suck, im not getting anythign apart from this and i cannot explain my professor, i need ot read a lot to understand the concept well, give me a clue as well guide me to the sites or books which helps me to learn and understand.

The FFM
The FFM's picture
The clue was right there and

The clue was right there and I don't want to just give you the answer
Look up "GC repeats"/"GC rich regions" (and think about hydrogen bonds) and also research loss of signal with repetitive DNA streches in relation to DNA sequencing. 

Biolink
Biolink's picture
 

 
GC-rich templates have unique secondary structures and melting temperatures and got three hydrogen bonds, which maintain a stronger association between the double-stranded DNA. DNA sequences with a high GC content as I understand will be relatively more difficult to denature and process, and may lead to a more challenging sequencing reaction. I understand this concept well now, well can you tell me what’s the size limit for base pairs which could be used for short gun sequencing. DNA sequencing can only be used for fairly short strands (100 to 1000 basepairs), longer sequences must be subdivided into smaller fragments, and subsequently re-assembled to give the overall sequence. Two principal methods are used for this: chromosome walking, which progresses through the entire strand, piece by piece, and shotgun sequencing, which is a faster but more complex process, and uses random fragments. I hope 1 MB is equal to 1,000,000 bp but here I have two options 1MB and 3MB as you say G and C content will be one difficulty aprt from that the size seems to be lesser compared to the other fungi, so on what basis I should select the fungal species now and will short gun method work here really.