ATS2458 Lecture Notes - Lecture 6: Northern Blot, Reverse Transcriptase, Gene Expression

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peachwolf588

20 Jan 2017

School

Monash University

Department

General Education Studies

Course

ATS2458

Professor

Asher Flynn

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Document Summary

The first step towards identifying and characterizing a gene is to isolate it from the rest of the genome and produce a large enough quantity of it to allow its investigation. Gene cloning is the process of isolating and amplifying a specific gene or dna fragment. The example here describes the basic steps involved in the conventional way of cloning a dna fragment using bacteria. In this case, the dna fragment containing the gene of interest was isolated from the entire genome by using specific enzymes, so called restriction enzymes. These are specialized enzymes that allow the site specific cutting of the dna at a particular sequence. The dna fragment is then inserted into a so called vector, something like a mini chromosome and introduced into bacteria. As the bacteria multiply the vector with the dna is also multiplied to produce many copies of this dna that can then be isolated in large quantity from the bacteria.

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

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

rubyhedgehog47

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.

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.

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

mauvecattle119

Recombinant DNA Technology

The questions all relate to the same topic and somewhat interrelate

1. Plasmids used in vitro to clone foreign DNA fragments are called ___

A. Transgenic.

B. Clones.

C. Vectors.

D. Conjugants.

2. VNTR alleles are hypervariable regions of human DNA that differ from each other in:

A. Location of internal sites recognized by restriction enzymes.

B. Variable number of point mutations.

C. Number of copies of an internally repeated DNA sequence.

D. Variable location on different chromosomes.

3. Probability calculations are used in forensic applications of DNA fingerprinting to determine if:

A. DNA from two different sources has matching alleles.

B. DNA samples were degraded before analysis.

C. A match between alleles of different DNA samples might have occurred by chance.

D. An unknown DNA sample is from a male or a female.

4. Plasmid vectors used in cloning often contain a gene for the N-terminal 146 amino acids of the enzyme Î²-galactosidase. What is the purpose of including this gene in the vector?

A. It allows selection of E. coli host cells that contain the plasmid.

B. It allows selection of E. coli host cells that contain plasmid in which the insert has been ligated.

C. It cleaves the insert to allow it to be ligated into the vector.

D. It enables the plasmid vector to replicate in E. coli host cells.

5.The first gene therapy clinical trial took place in 1990, on patients with SCID (severe combined immunodeficiency syndrome). Is this true or false?

A. True

B. False

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ATS2458 Lecture Notes - Lecture 6: Northern Blot, Reverse Transcriptase, Gene Expression

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