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BIOL 2303 Lecture Notes - Lecture 15: Restriction Fragment Length Polymorphism, Restriction Enzyme, Agarose

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pearferret848
27 May 2020
School
Carleton University
Department
Biology
Course
BIOL 2303
Professor
Ashkan Golshani

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Document Summary

The restriction enzymes recognize specific sequences of dna and it will digest it. Once you cut it, there will be spaces there so the long dna will become shorter dna strands. For ex: we know the given salmonella sequence and what kind of digestion we can get because we know the locations of the sequences, so we know we"ll get 3 strands. How can we see the strands, use eletroforese gel (agarose. ) and it gives a tiny hole that. The bands (cut dna strands ) are generated because of restriction digestions. If you don"t have enough dna to do rflp, then we do polymerase polymorphisms (diff morphs) (rflp) chain reaction (pcr) Pcr is when you use an enzymatic reaction (polymerase) to take template"s dna and amplify it. It"s done by taking one piece of double stranded dna (direction. Is very important), you need 2 primers, one of them binds to one strand,

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Related Questions

1.) A restriction recognition site is repeated 1 times on a circular DNA molecule. If you treat this DNA molecule with this restriction enzyme, how many DNA fragments will you get?

a. 1

b. 2

c. 3

d. Restriction enzyme does not cut a circular molecule

2.) In an agarose gel DNA electrophoresis, the DNA fragments are separated based on their

a. Size

b. Electrical charge

c. Nucleotides sequences

d. Density

e. All of the above

3.) DNA molecules are usually separated using agarose gel rather than polyacrylamide because:

a. Agarose polymerizes faster than acrylamide gel

b. Polyacrylamide gel has pore size larger than the size of DNA molecules

c. Agarose gel do not have pores on tis matrix and can hold large DNA molecules

d. Agarose gel has larger pore size similar to the size of DNA molecules

4.) Restriction endonucleases

a. Randomly recognize and cut DNA sequences

b. Hydrolysis the phosphodiester bond of DNA backbone

c. Recognize specific sequence on double stranded DNA

d. A and B

e. B and C

We have four DNA sequences that we need to digest (cut up), and have 6 restriction enzymes in stock in our lab. We want to find out which restriction enzyme we can use on each sample.

For each sequence, please indicate which restriction enzyme(s) would cut within each sequence. Each sequence may be cut by one, more than one, or no restriction enzymes.

Following the sequences are two questions. Please choose from the same list of enzymes to indicate the correct answer to these questions.

Restriction enzyme Digestion pattern
EcoRI

5' G|AATTC 3'

3' CTTAA|G 5'
HindIII

5' A|AGCTT 3'

3' TTCGA|A 5'
BamHI

5' G|GATCC 3'

3' CCTAG|G 5
AluI

5' AG|CT 3'

3' TC|GA 5'
SmaI

5' CCC|GGG 3'

3' GGG|CCC 5'
HbaI

5' GCG|C 3'

3' C|GCG 5'

(The | symbol indicates the specific cut sites.)

Each answer may be used once, more than once, or not at all.

1.

5' -TAGACTGAATTCAAGTCA- 3'

3' -ATCTGACTTAAGTTCAGT- 5'
2.

5' -ATACGCCCGGGTTCTAAA- 3'

3' -TATGCGGGCCCAAGATTT- 5'
3.

5' -CAGGATCGAAGCTTATGC- 3'

3' -GTCCTAGCTTCGAATACG- 5'
4.

5' -AATAGAATTCCGATCCGA- 3'

3' -TTATCTTAAGGCTAGGCT- 5'
5.

Which restriction enzyme(s) is(are) 4-cutters (i.e. they recognize and cut at 4-bp sequences, instead of 6-bp sequences)?
6.

Which restriction enzyme(s) do not leave a "sticky end" after cutting the DNA?
A.

Alu
B.

AluI, SmaI
C.

All 6 of these restriction enzymes.
D.

None of the 6 restriction enzymes listed.
E.

BamH1
F.

HindIII, AluI
G.

EcoR1
H.

SmaI
I.

SmaI and HbaI
J.

HbaI
K.

HindIII
L.

AluI, HbaI
M.

EcoRI, AluI
N.

AluI
O.

BamHI, AluI

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