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CSB349H1 Lecture Notes - Lecture 3: Euchromatin, Chromatin Remodeling, Repressor

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yellowcattle940
29 Mar 2017
School
UTSG
Department
Cell and Systems Biology
Course
CSB349H1
Professor
Alan Moses

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Document Summary

Genome sequencing: we determine the order of a, c, g, and t, dna sequencing technology has improved rapidly and the cost has also been reduced, the major technique being used today is sequencing by synthesis (illumine) They have special dna bases and when the bases join into a growing chain, they emit light and they make it so a, c, g, and t make different colour of light. They synthesize the dna and see what kinds of light is emitted. By recording the light, they can determine the sequence of letters. You can make a machine that can do this really quickly and in parallel. You can sequence millions of dna sequences at the same time. You ligate onto the sequences adaptors which are little bits of. Dna that will stick to a surface (silicon) that have primers. Adaptors will stick onto the primers and the dna fragments will attach to the surface.

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Related Questions

You decide to identify the CFTR mutation by analyzing the genomic DNA of your patients compared to healthy individuals. You specifically are looking to see whether a specific 3' gene truncation has occurred in the patients. You will determine this using hybridization techniques with samples from healthy and CF patients. Which of the following will allow you to accomplish this?

Using an RNA probe complementary to the region not removed by the truncation.

Using an RNA probe complementary to the region removed by the truncation.

Using an DNA probe complementary to the region not removed by the truncation.

Using an DNA probe complementary to the region removed by the truncation.

To conduct the hybridization experiment, you are trying to decide between using a DNA or RNA probe. Which would be ideal to use and why?

As both are composed of nucleic acids, using either would result in identical results.

An RNA probe because RNA has uracil bases.

An RNA probe because it could also be used in a translation experiment.

A DNA probe because it is more stable than RNA.

A DNA probe because RNA cannot bind to DNA.

Imagine Hershey/Chase had used an RNA virus (genome composed of RNA) instead of a DNA virus in their experiment. Would radioactivity still have been found in the pellet?

No, because only DNA can be labeled with radioactivity.

No, because the RNA genome would not enter the bacteria upon infection.

No, because while DNA and RNA nucleotides are similar, they are not identical.

Yes, because DNA and RNA nucleotides are similar.

Yes, because genome in any form (DNA, RNA, protein) would be labeled similarly.

The human genome consists mostly of non-coding DNA. Which of the following are benefits of this?

Random DNA mutations generally won't affect RNA and protein function.

It is faster to duplicate the genome when these are present.

The existence of introns can lead to multiple variations of proteins encoded by a single gene.

It is unlikely transposons would exist in the genome if there was too much protein coding DNA.

You accidentally add a mutant dNTP (which has an H instead of an OH connected to the 3’ carbon) to a reaction where DNA is being replicated. Which of the following is true of this mutant dNTP?

It can be incorporated into DNA strand but cannot form a phosphodiester bond with an incoming wild type dNTP

It can be incorporated into a DNA strand but cannot base pair with a complementary nucleotide

It can be incorporated into a DNA strand and can form a phosphodiester bond with an incoming dNTP, but only if it is another mutant dNTP

It cannot be incorporated into a DNA strand.

Andrew Murray's sister, Andrea, is adding to her brother's work on chromosomes. She is using cells that are unable to synthesize adenine (∆ade) and histidine (∆his). The plasmid she is currently working with consists of an origin of replication and the Ade gene.

Following her transformation of the plasmid into her yeast, what media will the cells be plated on to select for cells that have picked up the plasmid?

Media containing histidine

Media containing adenine

Media lacking adenine

Media lacking histidine

Andrew Murray's sister, Andrea, is adding to her brother's work on chromosomes. She is using cells that are unable to synthesize adenine (∆ade) and histidine (∆his). The plasmid she is currently working with consists of an origin of replication and the Ade gene.

She starts by attempting to add the centromere DNA into a plasmid containing the origin of replication. Unfortunately, when adding the centromere sequence, the origin of replication is removed, thus leaving a plasmid with only a centromere and selection marker. Following plasmid transformation, what growth result will she see on her plates?

No colonies

Little colonies

Big colonies

Andrew Murray's sister, Andrea, is adding to her brother's work on chromosomes. She is using cells that are unable to synthesize adenine (∆ade) and histidine (∆his). The plasmid she is currently working with consists of an origin of replication and the Ade gene.

She fixes the mistakes from the previous experiment and now has a complete plasmid (selection marker, origin of replication, centromere). She then inserts telomere sequences into the plasmid. How will this impact her transformation?

It will not impact her transformation

Her transformation will no longer work because plasmids don’t require telomeres

She will now see much larger colonies

She will now see fewer but larger colonies

She will now see smaller colonies

In the Meselson/Stahl experiment, E. coli were first grown in media containing heavy nitrogen, 15N, and then transferred to light nitrogen, 14N, at the beginning of the experiment.

Imagine that their data showed that replication occurs in a conservative manner instead of semi-conservative. What fraction of the DNA helices will consist of mixed DNA after 4 rounds of replication in this case?

None

More than 75%

25-50%

51-75%

Less than 25%

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.