BIOSC-101 Lecture Notes - Lecture 32: Dna Ligase, Genomic Library, Reverse Transcriptase

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azureprairie-dog22

31 Aug 2020

School

Diablo Valley College

Department

Biological Science

Course

BIOSC-101

Professor

wyler

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Document Summary

Steps to clone a gene of interest using a plasmid cloning vector and restriction. Isolate genomic dna containing the gene of interest and cut the genomic dna into fragments with a restriction enzyme. Cut a circular plasmid vector with the restriction enzyme and the restriction site for the enzyme is within the lacz+ gene. Combine the fragments with the cut plasmids, and seal with dna ligase; you get a mix of recombinant and nonrecombinant plasmids. Transform the plasmids into e. coli, some bacteria take up plasmids and some do not. While untransformed bacterium can"t grow on a medium containing ampicillin, transformed bacteria grow on the medium because of the ampr gene on the plasmid. Incubating the plate until colonies that contain the recombinant plasmids appear and are screened. The gene of interest has a unique dna sequence, and through dna hybridization it is identified when it base-pairs with a complementary dna/rna molecule called a nucleic acid probe.

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

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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

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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.

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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.

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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

A plasmid that carries two antibiotic resistance genes, to ampicillin and tetracycline, respectively, has one restriction site for EcoRI located within the tetracycline resistance gene. Eukaryotic DNA containing a gene of interest (g.o.i.) has been digested with diffferent restriction enzymes, none of which cut within the gene of interest sequence). The resultant fragments are added to plasmid DNA that has been digested with EcoR1 only; the mixture was then incubated with DNA ligase. Resulting plasmids (recombinant and otherwise) are added to competent bacteria lacking resistance genes and growing in nutrient broth. The bacteria are then plated and allowed to grow overnight producing isolated colonies. Samples of these colonies are then grown in four different media: nutrient broth plus ampicillin; nutrient broth plus tetracycline; nutrient broth plus ampicillin and tetracycline; and nutrient broth containing no antibiotics.

1. The bacteria containing the plasmid with the gene of interest successfully insertedin would grow in

A. Tthe nutrient broth only.

B. The nutrient broth, the ampicillin broth, and the tetracycline broth.

C. Tthe nutrient broth and the tetracycline broth only.

D. The ampicillin broth and the nutrient broth.E.Tthe ampicillin and tetracycline broth only.

2. Bacteria transformed with the plasmid alone (not containing any eukaryotic DNA), would grow

A. Only in the broth containing both antibiotics.

B. In the nutrient broth plus ampicillin, but not in the broth containing tetracycline.

C. In the broth containing tetracycline, but not in the broth containing ampicillin.

D. Only in the broth that contained no antibiotics.

E. In all four types of broth.

4. Bacteria that did not take up any plasmids at all would grow in which media?

A. The tetracycline and ampicillin broth only

B. The nutrient broth and the tetracycline broth only

C. The nutrient broth and the ampicillin broth only

D. The nutrient broth only

E. All four broths

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BIOSC-101 Lecture Notes - Lecture 32: Dna Ligase, Genomic Library, Reverse Transcriptase

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