You are attempting to clone the human insulin gene into E.Coli so that you can h

Question

You are attempting to clone the human insulin gene into E.Coli so that you can have access to unlimited human insulin for medical use. Insulin is produced in pacreas, but you have human skin cells to work with.

Design a probe around 16-20 bases for a primer based on the sequence provided. Be sure to clearly indicate where you come up with your probe. What is holding the two parts of this protein together? Be specific.

A chain 56i, ? 9 10111213141S 16 17 te ts ao 21 B chain 3456,1,10 "?13141516 17 1. 1, 20 GN 21 0 29 2 27 2 25 24 2 22

Explanation / Answer

A primer is a short nucleotide sequence of length 15 - 20 nucleotides which functions as the sites of DNA replication in a technique known as PCR (polymerase chain reaction). There are two primers Forward and Reverse. The primers will match the beginning and end of the DNA sequence that requires to be amplified, which in this case is the human insulin gene.

In this case let us take the forward primer.

The forward primer will be mostly complementary ( complementarity for nucleotides A-T, C-G) to the reverse strand at the front of the gene or in other words the beginning 15-30 nucleotides of the bottom strand of the DNA

So in order to design a probe for the primer, we need to first convert the amino acids into their complementary codons. Codons are triplets of nucleotides that when translated become amino acids that form the protei peptide chain.

Since the amino acid structure is given we will take the first ten amino acids and use that order to generate the complementary 30 nucleotides which will be used for the forward primer and by extension the site of the probe, which is what is needed for this question.

so the ten amino acids are glycine, isoleucine, Valine, glutamine, glutamine, cysteine, cysteine, threonine, serine and isoleucine

We can use multiple online tools or do the mapping manually with a codon chart available in any biochemistry text book

the aminoacids when converted to nucleotides look like this

5'-GGCATTGTCCAACAGTGTTGCACATCGATTTAA-3'

3'-CCGTAACAGGTTGTCACAACGTGTAGCTAAATT-5'

Whre the upper is the forward and the lower is the reverse strand

Now the forward primer is the complementary of the lower strand which means it will be similar to the upper strand, the probe has to bind or hybridise to the primer so it has to be complementary to the primer so it will be similar to the lower strand and complementary to the upper strand.

A probe is a single stranded nucleotide complementary to the target DNA so that ot binds to it and can be detected using a label built into the probe such as radioactive and fluorescent.

Since the length is mentioned as16-20 bases it is a oligonucleotide probe.

some of the steps in designing a oligonucleotide probe are

1) length between 18-50 since longer probes have higher hybridisation times and can form hairpin loops due to selg complementarity

2) the GC content should be 30-60% as it may cause non specific hybridiztion otherwise

3) Avoid complemtary regions

So based on these rules manually we can create a 16 nucleotide probe like so from the forward strand of the sequence complementary to the forward primer

5'-TAACAGGTTGTCACAA-3'

This has 37.5% GC content and no self complementarity with a length of 16 nucleotides.

This forms the basis of your probe

The probe is then labelled with biotin, it is chemically treated and attached to the DNA of the probe. Which then allows it to be detected with steptavidinafter the probe hybridizes with its target.

2) The A and B chain of the protein are held together by disulphide bonds between the cysteine residues at Chain A7-B7 and also chain A 20- B19 in the separate protein chains.

It is a covalent bond between two thiol groups in cysteine, and is one of the strongest bonds a protein can possess responsible partially among other things for the tertiary structure or functional confirmation of proteins.  

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