BIO206H5 Lecture Notes - Lecture 12: Start Codon, Point Mutation, Cysteine

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crimsonsnake651

1 Oct 2016

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The genetic code involved reading triplets; 4 different possibilities to code for the 20 amino acids; excess coding capacity. Comma-free; no spacing between one nucleotide to the next. 44 of the 64 combinations were potentially degenerate (amino acids specified by more than one codon) Codon: group of bases that specifies a single amino acid. The code is read in units of 3 nucleotides. 3 bases per codon (triplet) = 64 combinations. Synonyms: codons that specify the same amino acid. Degenerate: amino acids specified by more than one codon. E. g. cysteine has two codons, ugc & ugu. The standard genetic code: 61 of 64 codons encode amino acids, from 2-6 synonyms per amino acid, synonyms usually 3rd position wobble , 2nd position if more than 4 synonyms (e. g. leu, aug (met) is also start codon. A single point mutation could cause amino acid substitution e. g. agc ->aga causes ser ->arg. Degeneracy ->many mutations at 3rd codon position silent .

Related Questions

I need help with Part B. I believe that part A is correct.

a) You also wish to synthesize a degenerate oligonucleotide primer that could be used eventually to clone DNA that encodes your mutant protein. Design this degenerate oligo, keeping in mind the rules we talked about in class. More information about designing degenerate primers and primers in general are located on the following page.

Write out the sequence of this oligo here based on this amino acid sequence (Met Ala Pro Met Val Ala Glu):

My answer (Is it correct?)

I got:

5â AUG - (GCU/C/A/G) - (CCU/C/A/G) - (AUG) - (GUU/C/A/G) - (GCU/C/A/G) - (GAA/G) â3

3â UAC - (CGA/G/U/C) - (GGA/G/U/C) - (UAC) - (CAA/G/U/C) - (CGA/G/U/C) - (CUU/C) 5â

______________________________________________________________________________

(What I actually need help with and have no idea where to begin. Please help me)

b) If you synthesized the above oligonucleotide primer, how many different oligos would be present in the mixture?

Follow these rules Modified from the Thermo Fisher web site:

Designing degenerate primers:

Write out your amino acid sequence, label amino and carboxy termini

For each amino acid, use the genetic code to predict the various nucleic acid codons that can encode this amino acid. For all but Met, you should have redundancy present.

Now you need think about 5â and 3â considerations, and keep in mind that DNA within genes is double-stranded. It is helpful to keep in mind that the 5â end of a gene corresponds to the amino terminus of a protein. Using 5â and 3â labels write out a single strand DNA sequence corresponding to the your amino seq of interest. You may choose to start with only possible codon for each amino acid, or you can do all possibilities using the notation I showed you in class to account for the wobble position of codons.

Now make your DNA double stranded by filling in the opposite DNA strand; label the 5â and 3â ends of this second strand and make sure you have an anti-parallel arrangement of DNA strands.

If you havenât already, now consider the redundancy present on both strands, and make sure you have indicated sequences that account for all possible redundancies.

Realize that when a researched synthesizes a degenerate oligo that the final synthesis contains oligos with every substitution possible. The amount of degeneracy is defined by the number of different primer combinations in the mix. You can determine the degree of degeneracy by multiplying the number of changes present at each position together. If a position has no degeneracy then you multiple by 1 for that position.

Keep in mind that the trade-off between primer specificity and efficiency can be modified by altering the degeneracy of the primer. For example, the more degenerate the primers, the less specific annealing will be; however, decreased degeneracy will allow more potential to identify unknown variants. Anything over 1024 fold degeneracy is at the upper limit of potential usefulness.

Write out the sequence of this oligo here

b) If you synthesized the above oligonucleotide primer, how many different oligos would be present in the mixture?

RULES TO FOLLOW for creating primers (Modified from the Thermo Fisher web site):

Designing degenerate primers:

Write out your amino acid sequence, label amino and carboxy termini

For each amino acid, use the genetic code to predict the various nucleic acid codons that can encode this amino acid. For all but Met, you should have redundancy present.

Now make your DNA double stranded by filling in the opposite DNA strand; label the 5â and 3â ends of this second strand and make sure you have an anti-parallel arrangement of DNA strands.

If you havenât already, now consider the redundancy present on both strands, and make sure you have indicated sequences that account for all possible redundancies.

Designing Primers:

Good primer design is essential for a successful PCR reaction. There are many factors to take into account when designing the optimal primers for your gene of interest. This information is taken from the Fisher Scientific website and are used when actually designing an experiment. Here are some tips to consider when designing primers.

In general, a length of 18 nucleotides is the minimum necessary for efficient primer binding.

Try to make the melting temperature (Tmm) of the primers between 65Â°C and 75Â°C, and within 5Â°C of each other.

If the Tmm of your primer is very low, try to find a sequence with more GC content, or extend the length of the primer a little.

Aim for the GC content to be between 40 and 60%, with the 3' of a primer ending in C or G to promote binding. This is called a GC clamp.

Typically, 3 to 4 nucleotides are added 5â of the restriction enzyme site in the primer to allow for efficient cutting. Restriction enzymes are ENDO nucleases, so they cut better when their recognition site is not on the end of DNA.

Try to avoid regions of secondary structure, and have a balanced distribution of GC-rich and AT-rich domains.

Avoid intra-primer homology (more than 3 bases that complement within the primer) or inter-primer homology (forward and reverse primers having complementary sequences). These circumstances can lead to self-dimers or primer-dimers instead of annealing to the desired DNA sequences.

Its one problem with 7 questions

A) Most eukaryotic cells transcribe a relatively high portion of their genome, but most of those RNA transcripts are likely not encoding for proteins. Elucidating the function(s) of that "junk" RNA is one of the next big challenges in molecular cell biology. You isolate one of those transcripts and determine that its sequence consists of 50% C, 30% U, and 20% A. What is the percentage of Gs in this transcript? (don't write the unit)

B) Which stop codon(s) can be encoded by the RNA in the previous question ("How many times do we need to tell you to stop? 1").
List the codon(s) alphabetically. If you need to enter fewer codons than the alloted number of "blanks", enter N/A .

C) From what you can tell, the sequence of the transcript you isolated in the previous question* appears quite random. Assuming that it is random, you can expect to see the various combinations of any three nucleotides at a probability that depends of the frequency of each of the nucleotides. For example, you naturally should expect to see the triplet CCC more times than AAA. What is the expected percentage of triplets corresponding to stop codon(s) in this transcript (round to one decimal place and enter just the number without "%")? *(How many times do we need to tell you to stop? 1)

D) If the length of the transcript in the previous two questions is 2,500 nucleotides, how many stop codons do you expect to find in it, based on the frequency you calculated above?

E) On average, how many bases separate each of the occurrence of the stop codon(s) (round to the nearest integer).

F) The sequence between two potential stop codons is an "open reading frame" (ORF*). Upon closer examination of the RNA sequence from the previous questions** you determine the presence of an ORF that may encode for a putative protein which is 600 amino acid residues-long. How long is the ORF?

*Open reading frame: a stretch of DNA or RNA which is uninterrupted by a stop codon and may therefore encode for a protein.

**Questions "How many times do we need to tell you to stop" 1 through 5.

		27 bases
		1,800 bases
		2,500 bases
		259 bases
		600 bases
		83 bases
		200 bases

G) What may be the ramifications of your analyses of the RNA you isolated*? It is clearly containing an ORF which is longer than what you expected to find based on the sequence analysis you have conducted. What would be a logical conclusions based on the comparison of the lengths of the actual and predicted ORFs? Questions "How many times do we need to tell you to stop" 1 through 6. Pick 1, 2 or 3 below

		1) The difference between the actual and predicted sizes of the ORF is not big enough to be significant and therefore your initial hypothesis that sequence is random is clearly random, is supported.
		2) Nature is random and your results, using the Bard's words, may be "full of sound and fury" but are really "signifying nothing".
		3) The difference between the actual and predicted sizes of the ORF is rather big and may suggest that the sequence of the RNA may not be as random as it first appeared. This raises the hypothesis that what started as piece of "junk" RNA may be actually encoding for a protein.

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BIO206H5 Lecture Notes - Lecture 12: Start Codon, Point Mutation, Cysteine

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