MCB 2050 Lecture Notes - Lecture 3: Restriction Fragment Length Polymorphism, Restriction Map, Insertional Mutagenesis

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aquamarineimpala938

14 Jan 2017

School

University of Guelph

Department

Molecular and Cellular Biology

Course

MCB 2050

Professor

D Mosser

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Lecture 3: Techniques for DNA/RNA Analysis

Gel Electrophoresis

Electrophoresis uses an electric field to separate macromolecules in agarose (DNA/RNA) or polyacrylamide

(DNA or protein) gels

DNA and RNA Electrophoresis

Separation of a mixture of DNA (or RNA) fragments in porous gel matrix (agarose or polyacrylamide) in an

electric field

Since DNA is negatively charged, it will migrate toward the anode

(positive electrode)

Their rates of migration are inversely proportional to their sizes (i.e.

lengths – the shorter ones move faster than the longer ones)

Applications:

• Determine sizes of DNA fragments

• Isolate DNA fragments for cloning

• In preparation for Southern/Northern blotting

Steps of Electrophoresis (pic on right)

DNA Restriction Mapping

Restriction mapping – for physical mapping of DNA based on relative location of RE sites

DNA Restriction Mapping

Mappig of restritio sites of DNA A eaple of phsial appig

Applications:

• The relative positions of restriction sites on a DNA are used as markers. It provides an

overview of the organization of a DNA fragment, e.g. mapping the boundary of a gene or checking the

presence of a DNA insert

• The restriction sites are also used as genetic markers for diagnostics – RFLP (restriction fragment length

polymorphism)

• Can be used for cloning sub-fragments of the DNA and assembling a total genomic DNA sequence

Restriction Map of pBR322, an early bacterial cloning vector

1. What size fragments would be generated by digesting with Sal1 and Pst1?

3607 (Pst1) – 651 (Sal1) = 2956

4361-2956 = 1405

2. What would be the effect of cloning a foreign DNA fragment into the Pst1 site or

the Sal1 site?

Insertional inactivation of ampicillin or tetracycline resistance

Restriction Mapping: a linear DNA example

1. Where does Hindlll cut relative to EcoRI?

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Document Summary

Electrophoresis uses an electric field to separate macromolecules in agarose (dna/rna) or polyacrylamide (dna or protein) gels. Separation of a mixture of dna (or rna) fragments in porous gel matrix (agarose or polyacrylamide) in an electric field. Since dna is negatively charged, it will migrate toward the anode (positive electrode) Their rates of migration are inversely proportional to their sizes (i. e. lengths the shorter ones move faster than the longer ones) Restriction mapping for physical mapping of dna based on relative location of re sites. Mappi(cid:374)g of restri(cid:272)tio(cid:374) sites of dna (cid:894)a(cid:374) e(cid:454)a(cid:373)ple of (cid:862)ph(cid:455)si(cid:272)al (cid:373)appi(cid:374)g(cid:863) Applications: the relative positions of restriction sites on a dna are used as markers. If circular, the # of fragments equals the # of sites (ex. If linear, the number of fragments equals the # of sites +1 (ex. Determine if the dna is circular or linear.

Related Questions

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)?

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?

3 Gm

300 Kb

3 Mb

3 Gb

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

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MCB 2050 Lecture Notes - Lecture 3: Restriction Fragment Length Polymorphism, Restriction Map, Insertional Mutagenesis

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