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MGY299Y1 Lecture Notes - Deoxyribonuclease, Receiver Operating Characteristic, Encode

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School
UTSG
Department
Molecular Genetics and Microbiology
Course
MGY299Y1
Professor
Timothy Hughes

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Document Summary

Most of the functional dna appear to be regulatory elements. K562 a common cell line used in research. Rna-seq, et, cage are the most commonly used for transcript identification. Dnase-seq is the big one for chromatin accessibility identification. Long pieces of dna and just sequence the ends of them can then assume the middle part of the ends is transcribed. Also can know the size of the transcript. Take fragements and clone them into vectors have restriction sites (type 2, don"t cut at the site, it reaches over the site and cut) Rna-seq is more accurate for expression level: the above techniques are useful for identifying the location of sequence but not id the sequence. Dnases 1 can cut nucleosomal dna but can"t get into chromatin. Not against the rules to have nucleosomes in the empty space becuz dnase can cut nucleosomal dna.

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Related Questions

View/perform/read ALL THREE of the following prior to answeringthe questions.

http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/micro10.swf::Stepsin Cloning a Gene (Links to an external site.)

http://www.discoverbiotech.com/wiki/-/wiki/Main/Applications ofCloning (Links to an external site.)

http://www.wiley.com/college/boyer/0470003790/animations/cloning/cloning.htm(Links to an external site.)

Skip to question text.

From the list below, which of the following is the most logicalsequence of steps for splicing foreign DNA into a plasmid andinserting the plasmid into a bacterium?
I. Transformbacteria with recombinant DNA molecule
II. Cutthe plasmid DNA using restriction enzymes
III. Extractplasmid DNA from bacterial cells
IV. Hydrogen-bondthe plasmid DNA to nonplasmid DNA fragments
V. Useligase to seal plasmid DNA to nonplasmid DNA

IV, V, I, II, III
III, II, IV, V, I
III, IV, V, I, II
II, III, V, IV, I
I, II, IV, III, V

Flag this Question

Plasmids (or vectors) are important in biotechnology becausethey are

a vehicle for the insertion of recombinant DNA intobacteria.
surfaces for respiratory processes in bacteria.
recognition sites on recombinant DNA strands.
surfaces for protein synthesis in eukaryotic recombinants.
proviruses incorporated into the host DNA

Flag this Question

Plasmids are put into bacterial cells by

restriction enzymes
DNA ligase
binding of cohesive sticky ends
transformation

Flag this Question

Restriction enzymes usually

cut donor DNA evenly so smooth edges result
cut donor DNA but do not affect plasmids
make staggered cuts at specific sequences in DNA in both donorDNA and plasmid
are used in ligating plasmids into bacterial host cells
more than one of the above

Flag this Question

After combining DNA fragments in a cloning experiment, ___ isused to covalently join the DNA segments.

Restriction enzyme
DNA Ligase
Reverse transcriptase
DNA polymerase
Helicase

Flag this Question

It is theoretically possible for a gene from any organism tofunction in any other organism. Why is this possible?

All organisms have ribosomes.
All organisms have the same genetic code.
All organisms are made up of cells.
All organisms have similar nuclei.
All organisms have transfer RNA.

Flag this Question

Skip to question text.

Assume that you are trying to insert a gene into a plasmid andsomeone gives you a DNA sample cut with restriction enzyme X. Thegene you wish to insert from the given sample has sites on bothends for cutting by restriction enzyme Y. You have a plasmid with asingle site for Y, but not for X. Your strategy should be to

cut the plasmid with restriction enzyme X and insert thefragments cut with Y into the plasmid.
cut the plasmid with enzyme X and then insert the gene into theplasmid.
cut the DNA again with restriction enzyme Y and insert thesefragments into the plasmid cut with the same enzyme.
cut the plasmid twice with restriction enzyme Y and ligate thetwo fragments onto the ends of the human DNA fragments cut withrestriction enzyme X.
insert the fragments cut with X directly into the plasmidwithout cutting the plasmid.

Flag this Question

Which of the following is/are false in regard to expressionplasmids (also called expression vectors)?

They are used to make proteins using a cloned gene.
They contain a promotor.
They are the first plasmid type used to clone a gene.
They contain a terminator.
More than one of the above is false.

Flag this Question

What is NOT a potential problem(s) associated with usingbacteria containing a cloned eukaryotic gene (e.g. a human gene) toproduce a functional protein?

If the eukaryotic gene contains introns the bacteria will notremove them and the resulting amino acid sequence will be differentthat that made by a eukaryote.
The bacteria may not fold the protein correctly.
The bacteria may degrade the protein.
All of the above are potential problems.

Flag this Question

Cloning allows for production of proteins in much larger amountsthan occurs in the cells from which the gene is isolated.

True
False

Flag this Question

Question 111 pts

Gene cloning is used to do all of the following except

Make insulin
Making genetically identical animals (e.g. Dolly thesheep)
Make vaccines
Perform Gene Therapy
Making genetically engineered plants

Flag this Question

In your

View/perform/read ALL THREE of the following prior to answeringthe questions.

http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/micro10.swf::Stepsin Cloning a Gene (Links to an external site.)

http://www.discoverbiotech.com/wiki/-/wiki/Main/Applications ofCloning (Links to an external site.)

http://www.wiley.com/college/boyer/0470003790/animations/cloning/cloning.htm(Links to an external site.)

Skip to question text.

From the list below, which of the following is the most logicalsequence of steps for splicing foreign DNA into a plasmid andinserting the plasmid into a bacterium?
I. Transformbacteria with recombinant DNA molecule
II. Cutthe plasmid DNA using restriction enzymes
III. Extractplasmid DNA from bacterial cells
IV. Hydrogen-bondthe plasmid DNA to nonplasmid DNA fragments
V. Useligase to seal plasmid DNA to nonplasmid DNA

IV, V, I, II, III
III, II, IV, V, I
III, IV, V, I, II
II, III, V, IV, I
I, II, IV, III, V

Flag this Question

Plasmids (or vectors) are important in biotechnology becausethey are

a vehicle for the insertion ofrecombinant DNA into bacteria.
surfaces for respiratoryprocesses in bacteria.
recognition sites onrecombinant DNA strands.
surfaces for protein synthesisin eukaryotic recombinants.
proviruses incorporated intothe host DNA

Flag this Question

Plasmids are put into bacterial cells by

restriction enzymes
DNA ligase
binding of cohesive stickyends
transformation

Flag this Question

Restriction enzymes usually

cut donor DNA evenly so smoothedges result
cut donor DNA but do notaffect plasmids
make staggered cuts atspecific sequences in DNA in both donor DNA and plasmid
are used in ligating plasmidsinto bacterial host cells
more than one of theabove

Flag this Question

After combining DNA fragments in a cloning experiment, ___ isused to covalently join the DNA segments.

Restriction enzyme
DNA Ligase
Reverse transcriptase
DNA polymerase
Helicase

Flag this Question

It is theoretically possible for a gene from any organism tofunction in any other organism. Why is this possible?

All organisms haveribosomes.
All organisms have the samegenetic code.
All organisms are made up ofcells.
All organisms have similarnuclei.
All organisms have transferRNA.

Flag this Question

Skip to question text.

Assume that you are trying to insert a gene into a plasmid andsomeone gives you a DNA sample cut with restriction enzyme X. Thegene you wish to insert from the given sample has sites on bothends for cutting by restriction enzyme Y. You have a plasmid with asingle site for Y, but not for X. Your strategy should be to

cut the plasmid withrestriction enzyme X and insert the fragments cut with Y into theplasmid.
cut the plasmid with enzyme Xand then insert the gene into the plasmid.
cut the DNA again withrestriction enzyme Y and insert these fragments into the plasmidcut with the same enzyme.
cut the plasmid twice withrestriction enzyme Y and ligate the two fragments onto the ends ofthe human DNA fragments cut with restriction enzyme X.
insert the fragments cut withX directly into the plasmid without cutting the plasmid.

Flag this Question

Which of the following is/are false in regard to expressionplasmids (also called expression vectors)?

They are used to make proteinsusing a cloned gene.
They contain a promotor.
They are the first plasmidtype used to clone a gene.
They contain aterminator.
More than one of the above isfalse.

Flag this Question

What is NOT a potential problem(s) associated with usingbacteria containing a cloned eukaryotic gene (e.g. a human gene) toproduce a functional protein?

If the eukaryotic genecontains introns the bacteria will not remove them and theresulting amino acid sequence will be different that that made by aeukaryote.
The bacteria may not fold theprotein correctly.
The bacteria may degrade theprotein.
All of the above are potentialproblems.

Flag this Question

Cloning allows for production of proteins in much larger amountsthan occurs in the cells from which the gene is isolated.

True
False

Flag this Question

Question 111 pts

Gene cloning is used to do all of the following except

Make insulin
Making genetically identicalanimals (e.g. Dolly the sheep)
Make vaccines
Perform Gene Therapy
Making genetically engineeredplants

Flag this Question

In your

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.