🏷️ LIMITED TIME OFFER: GET 20% OFF GRADE+ YEARLY SUBSCRIPTION →
Log in
HomeClass Notes1,200,000CA670,000

Biochemistry 2288A Lecture Notes - Lecture 5: Dna Sequencing, Rna-Seq, Dna Ligase

21 views19 pages
School
Western University
Department
Biochemistry
Course
Biochemistry 2288A
Professor
Susan Meakin

For unlimited access to Class Notes, a Class+ subscription is required.

Document Summary

Cloning of yfg cont (last class we made cdna from a tissue that expressed yfg) then pcr to amplify the cdna, run on agarose gel to purify it, now we want to clone it. To get yfg into e. coli, we first insert it into a plasmid. Plasmid: a fragment of dna that replicates independently from the host chromosome. E. coli chrms: one copy, 5 million base pairs. Plasmid dna: copy # varies depending on the plasmid from 1-100 copies/cell. A way to break up dna into defined, workable units. Restriction ez are used to get yfg into the plasmid. Restriction ez cut dn into defined workable units. Site specific dna binding proteins that recognize and cleave palindromic dna seq. usually of 4, 6, or 8 base pairs. Note: dbl stranded dna is often written only indicating onle one strand from 5"-3". Isolated from bacteria where their native role is to act as anti viral agents.

Get access

Grade+20% off
$8 USD/m$10 USD/m
Billed $96 USD annually
Grade+
Homework Help
Study Guides
Textbook Solutions
Class Notes
Textbook Notes
Booster Class
40 Verified Answers
Class+
$8 USD/m
Billed $96 USD annually
Class+
Homework Help
Study Guides
Textbook Solutions
Class Notes
Textbook Notes
Booster Class
30 Verified Answers

Related Documents

Biochemistry 2288A Lecture Notes - Lecture 14: Apoptosis, Paclitaxel, Cell Adhesion
tantermite285
Biochemistry 2288A Lecture Notes - Lecture 22: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Gel Electrophoresis
viridiancricket542
Biochemistry 2280A Lecture : Restriction Enzymes are Enzymes
orchidotter238

Related Questions

1. We can construct our gene library by cloning the PstI fragments into either plasmid pBR322 or phage lambda. Which one should we choose and why?

2. Suppose we want to insert the PstI fragments into the Bam HI site within the gene for tetracycline resistance (tetr) in plasmid pBR322. If both of these restriction enzymes produce cohesive ends, how can this be done?

3. After transforming E.coli with the plasmids, how can we identify the cells that contain a chimeric plasmid (containing DNA insert)?

4. If a million tetracycline-sensitive, ampicillin-resistance clones are grown on nutrients agar plates, how are we going to detect the rare clone or clones that carry the gene for protein P?

5. After selecting a clone carrying the gene for protein P, its ells are propagated to high density in nutrient broth. The chimeric plasmid (with an insert of the gene for protein P) is then extracted and purified from the rest of the cellular DNA. How can we now isolate the gene from the plasmid?

6. How can we demonstrate that the gene we have isolate is indeed the one for protein P?

Which of the following did Erwin Chargaff observe?

A)

Base composition changes as the organism ages.

B)

Base composition of DNA does not vary from one species toanother.

C)

In all species the number of adenines equals the number ofguanines.

D)

In all species A + T = G + C.

E)

DNAs isolated from different tissues of the same species havethe same base composition.

The melting temperature (Tm) of a DNA duplex is:

A)

the temperature at which double-stranded nucleic acids degradeinto free nucleotides.

B)

the temperature at which double-stranded DNA is toosingle-stranded to ever renature back to double strands.

C)

the temperature at which double-stranded DNA is completelymelted.

D)

the temperature at which double-stranded DNA has become 50%single-stranded.

E)

the temperature at which double-stranded DNA begins to melt.

Given your knowledge and the descriptions of each of thesemethods, which of the following does NOT rely on the ability ofnucleic acids to hybridize with each other?

A)

Southern blotting for the detection of specific DNA fragmentsthat have been separated by gel electrophoresis.

B)

Colony blot hybridization to identify a specific sequence from acollection of sequences that have been inserted into bacteria.

C)

Northern blotting for the detection of specific RNA fragmentsthat have been separated by gel electrophoresis.

D)

A DNA oligonucleotide microarray incubated with probes made fromcDNA.

E)

Western blotting for the detection of specific proteins thathave been separated by gel electrophoresis.

Which of the following is a possible reason why DNA uses thymineinstead of uracil?

A)

Cytidine is deaminated to uracil so it would be difficult forany repair mechanism to differentiate between uracil that belongsin the sequence and uracil that resulted from deamination.

B)

Adenine is deaminated to uracil so it would be difficult for anyrepair mechanism to differentiate between uracil that belongs inthe sequence and uracil that resulted from deamination.

C)

When uracil is bound to a deoxyribose it will become morereactive, forming covalent bonds with other bases.

D)

None of the above.

Which of the following is an enzyme used in cloning to breakcovalent bonds?

A)

DNA ligase

B)

Restriction endonuclease

C)

Reverse transcriptase

D)

DNA polymerase I

E)

Alkaline phosphatase

Which of the following could you use to remove the 5' phosphatesafter cleavage of a plasmid with a single type of restrictionenzyme to ensure that the plasmid would not simply fuse backtogether upon addition of ligase?

A)

Reverse transcriptase

B)

Alkaline phosphatase

C)

DNA ligase

D)

DNA polymerase I

E)

Restriction endonuclease

Which of the following would you use to create a cDNA librarythat you would not use for a genomic library?

A)

Alkaline phosphatase

B)

Reverse transcriptase

C)

Restriction endonuclease

D)

DNA ligase

E)

DNA polymerase I

A plasmid vector typically has which of the followingfeatures?

A)

An origin of replication

B)

A selectable marker to ensure that the only bacteria growingcontain the intact or recombinant vector.

C)

Unique restriction sites for insertion of DNA.

D)

Small size to facilitate entry into the cell and biochemicalmanipulation of the DNA.

E)

All of the above are features of plasmid vectors.

Which of the following is FALSE of genomic or cDNAlibraries?

A)

Genes represented in cDNA libraries include control sequencesthat direct transcription.

B)

cDNA libraries contain cDNAs made from mRNA.

C)

Genomic libraries contain fragments of genomic DNA that aregenerated by partial digests with restriction enzymes.

D)

Genomic libraries usually contain large fragments and are likelyto be constructed in YAC or BAC vectors.

E)

cDNA libraries contain sequences from expressed genes only.

Which of the following is NOT a typical component of apolymerase chain reaction (PCR)?

A)

dNTPs

B)

DNA containing the sequences to be amplified

C)

DNA ligase to connect the fragments together

D)

Primers complementary to each end of the sequence to beamplified

E)

Heat stable Taq polymerase

How can a fusion protein be formed?

A)

Site-directed mutagenesis where a fragment is replaced with asynthetic sequence containing the altered DNA sequence.

B)

Two proteins can recombine in the bacterial cell to form a newhybrid protein.

C)

Portions of two different genes are ligated together to create anew hybrid gene. When expressed the gene product is a fusion of thetwo gene products.

D)

Using oligonucleotide-directed mutagenesis to introduce specificmutations that cause the expressed protein to fuse to otherproteins.

E)

A portion of the gene can be removed to form a shorter gene andtherefore a shorter protein.

Why is green fluorescent protein (GFP) so useful in visualizingfusion proteins in eukaryotes?

A)

The GFP protein recognizes and binds to fusion proteins allowingthe location of the fusion protein to be determined.

B)

The reaction is only dependent on the presence of GFP and oneother cofactor.

C)

It is easily cloned and expressed in bacterial cells.

D)

The presence of just a few molecules of GFP fusion proteins canbe sufficient for observing the proteins microscopically allowingthe study of its location and movements in the cell.

E)

It is derived from fire flies, which are easy to cultivate inthe lab.

You wish to determine the cellular location of a protein butunfortunately it is not particularly antigenic (can t develop astrong antibody response). How could you solve this problem?

A)

Create a fusion protein with an epitope tag that can bevisualized by incubation with fluorescently tagged antibody to theepitope.

B)

Create a fusion with green fluorescent protein (GFP).

C)

Express the protein as a fusion with biotin. The addition of GFPwill cause fluorescence.

D)

A and B might work.

E)

A, B, and C might work.

F)

None of the above will work.

A positive result for the yeast two-hybrid analysis means thefollowing:

A)

The proteins that you are studying can be made into fusionproteins, but do NOT interact with each other within the nucleus ofyeast.

B)

A fusion protein containing the Gal4p activation domain, hasbound to RNA polymerase resulting increase transcription ofnumerous genes, including the reporter gene, and formation ofcolonies.

C)

Two fusion proteins, one containing the Gal4p binding sitedomain and one containing the Gal4p activation domain, haveinteracted through their Gal4p domains resulting in transcriptionof the reporter gene.

D)

Two fusion proteins interact with each other and because of theGal4p binding site domain and the Gal4p activation domain containedin each fusion protein, the reporter genes are activated and thecells die resulting in no colonies.

E)

The colonies growing are expressing the two fusion proteins, butthe two proteins have not interacted with each other.

Which of the following is the approximate size of the humangenome?

A)

3 Gm

B)

300 Kb

C)

3 Mb

D)

3 Gb

E)

3 Mm

Which of the following is correct about the structure orlocation of genes?

Question 22 options:

A)

Prokaryotic genes typically possess their own regulatorysequences that only control expression of a single gene.

B)

Eukaryotic genes often encode introns that are spliced outbefore translation.

C)

Eukaryotic genes are mostly located at the telomeres and thecentromeres of chromosomes.

D)

Eukaryotic genes are typically arranged in operons.

E)

Prokaryotic genes often encode exons that are spliced out beforetranslation

Directions: Write out your answers in the word document and then copy and paste everything into the text box. If you do not include the question along with your answer, you will be marked down.

1. What would happen if you put pGLO directly onto an agar plate and you checked on it after letting it incubate?

a. It would glow

b. It would grow

c. Nothing

d. E.Coli would grow

2. Explain in a short sentence or two your reasoning for question 1.

3. What shape is a plasmid?

4. How is a plasmid different from human DNA? Write one difference.

5. Bacteria can share DNA through a unique method called transformation. What are the TWO requirements a bacteria cell must possess in order to share DNA with another bacteria cell?

6. Why during our experiment did we put our e.Coli with pGLO inserted in it onto agar plates with ampicillin (Amp)?

a. So that it would kill the e.Coli and serve as our control

b. To kill all the e.Coli that did not take up the pGLO

c. To create antibiotic resistant bacteria

d. To cause the e.Coli to glow

7. Yeast cells are what type of cell?

a. Bacteria

b. Eukaryotes

c. Prokaryotes

d. Archaea

8. Why would someone study yeast cells instead of human cells?

9. Do you agree or disagree with the following statement: If your cells is exposed to UV light and it causes a thymidine dimer, your cell is doomed to die.

10. Are wildtype (control) yeast immune to UV light damage?

11. BTB was a dye we used to directly measure ________

a. PH

b. Oxygen

c. Carbon Dioxide

d. Carbon

12. BTB was a dye we used to INDIRECTLY measure ________

a. PH

b. Oxygen

c. Carbon Dioxide

d. Carbon

13. What is one reason why a plant would want to close its stomata?

14. What is one function that ground tissues provides for a plant?

15. List one difference between monocots and dicots

16. Why is it important for a plant to have stomata? Write one reason why

17. What is the purpose of xylem in a plant cell?

18. Pholem is also important to plant cells, what is it’s purpose?

19. This theory explains why trees can grow to such tall heights and still provide water to their leaves.

a. Translocation

b. Transpiration

c. Cohesion-Tension

d. Vacuum

20. Fill in the blank

Pholem is considered to be a(n) _________ transport system while xylem is considered a(n) _______________transport system.

21. Apical meristems are found in these two places on a plant

a. At the site of new leaf growth and the roots closes to the top of the soil

b. Vascular tissue and ground tissue

c. Ground tissue and dermal tissue

d. The point of new growth on the stem and root

22. Fill in the blank.

Xylem moves substances _____ while phloem moves substances _____ a plant.

23. What are the two types of digestive mechanisms?

24. What is one benefit that mastication has for digestion?

25. What is an example of an organism that digests food intracellularly?

26. What is an example of an organism that digests food extracellularly?

27. Give two examples of where surface area is increased in a small space to aid in absorption within the human body

28. Name one difference between arteries and veins.

29. What is the role of capillaries in the circulatory system?

30. How are the terms “excretion” and “defecation” different?

31. What is the role of the ADH hormone?

32. What does the term “Medulla” refer to when talking about mammalian tissue?

a. The outer edges of a tissue

b. The middle of a tissue

c. The top of a tissue

d. The bottom of a tissue

33. Fill in the blank

Mammals have a _______ circulatory system while insects have a _______ circulatory system.

34. What is one benefit a closed circulatory system provides?

35. What is hemolymph?