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

Biology 1202B Lecture Notes - Lecture 1: Genomic Library, Complementary Dna, Reverse Transcriptase

13 views2 pages
pinkant517
15 Jun 2016
School
Western University
Department
Biology
Course
Biology 1202B
Professor
Ian Mac Donald

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

Document Summary

A collection of clones that contains a copy of every dna sequence in a genome is called a genomic library. To convert single stranded mrna to double stranded dna for clonging first the researchers use the enzyme reverse transcriptase to make a single stranded dna that is complementary to the mrna. Mran strand with an enzyme and use dna polymerase to make a second. Dna strand that is complementary to the first. After dding restriction site to each end, researchers insert the cdna into a cloning vector described for the genomic library. The genes in eukaryotic nuclear dna typically contain many introns, spacer sequences that interrupt the amino acid coding sequence of a gene. Because bacterial dna does not contain introns, bacteria are not equipped to process eukaryotic mra already has the introns removed, so bacteria can transcribe and translate it accurately to make eukaryotic proteins.

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

Biology 1202B
Final Exam
Study Guide
Biology 1202B Study Guide - Midterm Guide: Restriction Enzyme, Genomic Library, Hybridization Probe
erinfox426
Biology 1202B Chapter Notes - Chapter 15: Southern Blot, Filter Paper, Intron
bluetermite858
Biology 1202B Lecture Notes - Lecture 16: Kary Mullis, Microsatellite, Dna Profiling
blushcrocodile900

Related Questions

Are the following statements true or false? Please explain your answers.

1. Digestion of genomic DNA with AluI, a restriction enzyme that recognizes a four-nucleotide sequence, produces fragments that are all exactly 256 nucleotides in length.

2. To make a cDNA library, both a DNA polymerase and a reverse transcriptase must be used.

3. It is possible for a coding region of a gene to be present in a genomic library prepared from a particular tissue but to be absent from a cDNA library prepared from the same tissue.

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

10. I am interested in cloning the "hox" gene in my favorite non-model organism - clam shrimp. I make a cDNA plasmid library from adult females, plate it, and Sanger sequence 60,000 random clones. Since clam shrimp likely only have about 20,000 genes, I expect to sequence the hox gene roughly 3X. Is this a reasonable research strategy?

A. No, a cDNA library will not contain hox genes, they are non-transcribed DNA elements B. Yes. The probability of getting the hox gene is pretty high given that you have 3X

sequencing coverage of the library.
C. No, the probability of any given clone being a hox gene is NOT 1/20,000. It is

proportional to the expression level of the gene (and that could be low).
D. No, the probability of getting a hox gene is proportional to its length, and hox genes are

short

11. If I said I was going to sequence your genome using an "assembly by reference" approach, what does this mean?

A. I would make an Illumina gDNA library of your genome, Illumina sequence it, and assemble your genome using sequence overlaps

B. I would make a plasmid gDNA library of your genome, Sanger sequence it, and assemble your genome using sequence overlaps

C. I would make an Illumina cDNA library of your genome, Illumina sequence it, and identify mutations in promoters

D. I would make an Illumina gDNA library of your genome, Illumina sequence it, and identify variants by aligning the reads to a "reference" human genome

Explanation would be greatly appreciated! Final in two hours so plez help