MCDB 427 Lecture Notes - Lecture 3: Molecular Cloning, Affinity Chromatography, Peptide

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purplebeaver856

21 Dec 2016

School

U of M

Department

Molecular, Cellular and Developmental Biology

Course

MCDB 427

Professor

Janine Maddock

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Document Summary

We go through many cycles which each involve 3 subsets that are all related to change in temperature. Redo and check the answer to the question of the day (previous lecture) Another advantage is the small amount of dna that is needed. Compared to cloning in a cell, it"s about (cid:883)(cid:882)-100x faster. Essentially, it"s a process of just changing temperature so it"s done in a fancy heating. )f you don"t have any sequence information for the target dna of interest, there"s. Another disadvantage is that it"s easily contaminated due to the fact that you only element machine. The biggest one is that you need some sequence information in order to design the oligonucleotide primers which are the bases of where the amplification will occur need a small amount of starting material. Pcr has been used in all sorts of ways. We will focus on how it was used for dna cloning and study gene expression.

Related Questions

The success of PCR amplification depends on many factors, one being that the primers need to be in a large excess to the template DNA to drive the reaction forward over many cycles. However, looking at the reaction components, the primers (125 ng) are used at an even higher mass amount than the template DNA (100 ng). Explain. Hint: The mass amount is deceiving. You can calculate the ratio another way.

Sorry this is the only information we were given. Like I dont even know how to answer this :(

peachprairie-dog223

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.

(tautomerization) The most common form of guanine is the ketoform, not the enol form.

True

False

What term describes the allowed degree of base pair matchingduring annealing, varying from lots of base-pair mismatches allowedto allowing only perfect base-pair matching.

stringency

aneuploidy

polyploidy

quantitative PCR

restriction fragment length polymorphism

I have an individual with a disease we will call SSI-itis,brought on by extreme exposure to cannabis. I do a southern andfind the gene affected. Another of his classmates exhibits theexact same symptoms, but that gene in hiw is wt. A southern in thesecond student finds a mutation in another gene. This is an exampleof

pleitropy

allelic heterogeneity

locus heterogeneity

dominant traits

recessive traits

penetrance

If a person carrying a dominant allele does not demonstrate thephenotype strongly, but still differs from wt, thisdemonstrates?

haploinsufficiency

penetrance

expressivity

monogenic traits

polygenic traits

I find three alleles for a gene in a fruit fly. One allelecreates the ability for the fly to eat graphite. Another allelecreates the ability for the fly to eat coal. The third alleleallows the fly to eat diamond. These alleles are all dominant, andwt cannot eat any of these substances. Which statement below is themost true?

Upon mating a bunch of the flies carrying these alleles, Ishould not find a fly that can eat graphite, coal, and diamond.

I would consider the ability to eat these substances an exampleof pleitropy because they are all carbon.

I would expect the ability to eat diamond to be dominant to theability to eat graphite.

These flies are demonstrating locusheterogeneity.

Expression vectors in prokaryotes do not make functionaleukaryotic gene products in bacteria very well because

the codon sequence for prokaryotes is different than the codonsequence in eukaryotes

there are no disulfide bridges formed in proteins normally madein prokaryotes

prokaryotic expression vectors cannot translate eukaryticsequences

eukaryotic genes have introns, and prokaryotes don't

eukaryotic genes have exons and prokaryotes don't

Which of the following is not used in PCR amplification ofDNA?

Double-stranded DNA template

Oligonucleotide primers

DNA polymerase

DNA ligase

ddNTPs

I create a knockout mouse using the agouti/black fur systemdiscussed in the online lecture and in the book. When I cross theagouti offspring of the originally engineered mouse, I find a ratioof 2 agouti mice to 1 black furred mouse. What is the bestexplanation?

The gene knocked out was recessive.

The gene knocked out was dominant.

The gene knocked out was a lethal gene.

The knockput was integrated into a random spot, and did notknockput the original gene.

A loss of function mutation is usually recessive, but canactually be a dominant mutation because of what effect?

haploinsufficiency

dominant negative mutations

allelic heterogeneity

locus heterogeneity

pleitropy

A genetic carrier of a disease can have a form of the diseasethat only shows intermittent symptoms, while the full homozygousrecessiuve individual would show all of the symptoms of thedisease.

True

False

Wild type, or wt, refers to the genotype that is the typicalgenotype found in nature. Now read that again. Once more. Nowanswer.

Answer

True

False

mauvekangaroo793

MCDB 427 Lecture Notes - Lecture 3: Molecular Cloning, Affinity Chromatography, Peptide

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