BIO SCI 98 Lecture Notes - Lecture 4: Ribosome, Valine, Acylation

Double check to see if I got these right? 3 questions

Some Processes Involved in DNA Replication

1.The replication origin is identified.

2 DNA primase builds RNA primer.

3Okazaki fragments are spliced by DNA ligase.

4Helicases bind and uncoil the DNA double helix.

5 RNA primer is the starting point for DNA polymerase.

6 DNA replication ends at the telomere where DNA codes fortermination.

7 DNA polymerase adds complementary nucleotides in the 5' to 3'direction in short segments called Okazaki fragments.

The sequence in which the events numbered above occur during DNAreplication of the lagging strand: Number 1 - 7 in order.

I put: 1,4,2,5,7,3,6

__________________Question 2:

Some Functions of Enzymes

1. Build DNA strands
2. Build RNA primers
3. Build mRNA strand
4. Cleave DNA strands
5. Splice Okazaki fragments

Enzyme:
Helicase :4
DNA ligase :5
RNA primase :1
RNA polymerase :3
DNA polymerase:2

Question 3:

like mRNA, tRNA has a ribose sugar, U instead of T, and issingle stranded. Unlike mRNA, which remains a long single strand ofnucleotides, tRNA folds so that some areas pair up.

The resulting structure has an anticodon on one end and a site foran amino acid to attach on the other end. There is basecomplementarity (A pairs with U and G pairs with C) between an mRNAcodon and tRNA anticodon.

If the amino acid tyrosine attaches to a tRNA, which of thefollowing anticodons could be at the opposite end of the tRNAmolecule?

Select one:

a. ATA and ATG

b. UAU and UAC

c. AUA and AUG

d. AGA and AGU

I picked B.

2

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

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 you need think about 5’ and 3’ considerations, and keep in mind that DNA within genes is double-stranded. It is helpful to keep in mind that the 5’ end of a gene corresponds to the amino terminus of a protein. Using 5’ and 3’ 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 5’ and 3’ ends of this second strand and make sure you have an anti-parallel arrangement of DNA strands.

If you haven’t 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.

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 65°C and 75°C, and within 5°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 5’ 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.

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

4.

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:

5’ AUG - (GCU/C/A/G) - (CCU/C/A/G) - (AUG) - (GUU/C/A/G) - (GCU/C/A/G) - (GAA/G) ‘3

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

______________________________________________________________________________

______________________________________________________________________________

(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 5’ and 3’ considerations, and keep in mind that DNA within genes is double-stranded. It is helpful to keep in mind that the 5’ end of a gene corresponds to the amino terminus of a protein. Using 5’ and 3’ 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 5’ and 3’ ends of this second strand and make sure you have an anti-parallel arrangement of DNA strands.

If you haven’t 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.