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:

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.

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 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.

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.

QUESTION 1

A mutation caused by exposure to gamma rays is called a spontaneous mutation

	a.	True
	b.	False
	c.	Sometimes, depending on the type of base change
	d.	Gamma rays do not cause mutations

QUESTION 2

A recessive mutation affecting an essential biochemical pathway can be detected________

	a.	when the organism is heterozygous for that mutation.
	b.	when the organism is homozygous for that mutation.
	c.	in either homozygous or heterozygous conditions.
	d.	only by sequencing (recessive mutations never show a phenotype).

QUESTION 3

During DNA replication, the newly synthesized chain grows by adding

	a.	nitrogenous bases
	b.	the sugar-phosphate backbone
	c.	nucleosides
	d.	dNTPs

QUESTION 4

During replication, the DNA strand 5â- AAGTCTAGCCTAG -3â will serve as a template for the polymerization of:

	a.	5â- CTAGGCTAGACTT -3â
	b.	5â - TTCAGATCGGATC -3â
	c.	5â - GATCCGATCTGAA -3â
	d.	3â - AAGTCTAGCCTAG -5â

QUESTIOn 5

Given the following DNA sequences, select which statement(s) is(are) correct.

Sequence 1:

5â-TGGACGCTAA-3â

3â-ACCTGCGATT-5â

Sequence 2:

5â-AATCGCAGGT-3â

3â-TTAGCGTCCA-5â

Sequence 3:

5â-ACCTGCGATT-3â

3â-TGGACGCTAA-5â

	a.	Sequences 1 and 2 are the same
	b.	Sequences 1 and 3 are the same
	c.	Sequences 2 and 3 are the same
	d.	Sequences 1, 2 and 3 are all different

QUESTION 6

Repetitive sequences in the genome are hotspots for:

	a.	Deamination
	b.	Depurination
	c.	Thymine dimer formation
	d.	Replication errors

QUESTION 7

Select which statement(s) about the Ames test is(are) correct

	a.	it has been designed to understand mutation repair systems in Salmonella typhimurium
	b.	it uses mammalian liver extract
	c.	it allows to study whether chemical compounds or their enzymatic breakdown products are mutagenic
	d.	it is based on whether a chemical compound causes reversion mutations from his+ to his-
	e.	two of the above are correct
	f.	three of the above are correct

QUESTION 8

Select which statement(s) is(are) correct.

	a.	All DNA strands have a direction, and it is specified by the carbons in the sugar backbone.
	b.	All DNA strands have a direction, and it is specified by hydrogen bonds between nucleotides.
	c.	In order to form proper base pairs in a double stranded DNA molecule, the two strands must run in opposite directions.
	d.	A and C are correct.
	e.	B and C are correct.

QUESTION 9

Studies of gene mutation frequencies have shown that:

	a.	mutations are rare, and genomes are generally stable.
	b.	mutation frequencies differ among organisms and also between genes, suggesting certain genes are more susceptible to mutation.
	c.	mutation frequencies are consistent between organisms, and each region of DNA is equally susceptible to random mutations.
	d.	Both A and B are correct.
	e.	Both A and C are correct.

QUESTION 10

The compound 5-bromodeoxyuridine (BrdU) is a derivative of uracil, and if BrdU becomes incorporated during DNA replication, it pairs with adenine. This compound is best classified as which type of mutagen?

	a.	base analog
	b.	base inducer
	c.	intercalating agent
	d.	oxidative agent
	e.	alkylating agent

QUESTION 11

The rate of mutation of the fruit fly is higher than the rate of mutation of Algae

True

False

QUESTION 12

Thymine dimers are most commonly caused by which of the following?

	a.	X-rays
	b.	Alkylating agents
	c.	U.V. irradiation
	d.	DNA intercalating agents

QUESTION 13

What chemical group is found in the 3â end of a DNA strand?

	a.	an alcohol
	b.	a hydroxyl
	c.	a methyl
	d.	a phosphate

QUESTION 14

What chemical group is found in the 5â end of a DNA strand?

	a.	an alcohol
	b.	a hydroxyl
	c.	a methyl
	d.	a phosphate

QUESTION 15

Which is the correct order of molecules binding to DNA during DNA replication?

	a.	Helicase, SSB, primase, DNA pol III, DNA pol I, ligase
	b.	SSB, DNA pol I, ligase, helicase, DNA pol I, primase
	c.	primase, helicase, DNA pol III, ligase, DNA pol I, SSB
	d.	SSB, helicase, primase, DNA pol III, DNA pol I, ligase

QUESTION 16

Which statement(s) is(are) correct about strand slippage?

	a.	It can cause disorders such as Huntingon disease.
	b.	It is a process that causes mutations altering the number of DNA repeats.
	c.	It is a process that incorporates nucleotide base analogs and trinucleotide repeats.
	d.	a and b are correct.
	e.	b and c are correct.
	f.	a, b and c are correct.

help needed asap.

chocolatesea-lion434

Biology 1002B Lecture Notes - Lecture 21: Hbb, Gene Duplication, Alternative Splicing

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