1. You are sequencing a 350bp lizard gene using the Sanger dideoxy sequencing me
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Question
1. You are sequencing a 350bp lizard gene using the Sanger dideoxy sequencing method. You have amplified the region using the primer 5'-ATCCGGGCG-3'. What will be the length of the lowest band in your sequencing gel?
2. Which of the following answer(s) are true (select all that apply)?
Question options:
PCR results in exclusively the desired region to be amplified.
PCR primers must be outside the region of interest. PCR primers must be flanking the region of interest.
Sequencing primers must be flanking the region of interest.
All of the nucleotides used in Sanger Sequencing would also be able to be used for a PCR reaction where you are trying to amplify a gene of interest.
Sanger sequencing requires dNTPs.
A standard PCR reaction requires ddNTPs Small DNA fragments on a gel would be closer to the wells on a gel than larger DNA fragments.
Explanation / Answer
1. The lenghth of the lowest band in the sequencing gel is 3'- atccgggcg-5'.
2. PCR results in exclusively the desired region to be amplified.
PCR primerss must be inside the region of interest. a primer should be specific to your gene of interest only. if should bot bind outside the gene otherwise a false result will obtained on agarose gel electrophorosis run after the PCR step.
if PCR primers from dimers with each other, they cant bind to templete reducing the ability to copy the template and exponentially amplify the DNA lower yield of desired product.
by taking AtABCG40 gene from arabidopsis as my referenve gene, it will blast it with oil plam database. later you will get full sequence of the gene.
The DNA sample is divided into four separate sequencing reactions, containing all four of the standard dNTP's the DNA polymerase, the only one of the four ddNTP's for each reaction.
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