BIO374H5 Lecture Notes - Lecture 3: Western Blot, Agarose Gel Electrophoresis, Northern Blot

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brownhornet249

2 Nov 2017

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Gene cloning is different from animal cloning; gene cloning is copying a particular gene and not the whole animal. Tools required for recombinant dna: restrictive enzymes is a scissors that do the cutting of the gene. They cut the phosphodiester bond (bond between the nucleotides) The sequence they are cutting appears to be the same forward and backwards. Cohesive ends are better for biotechnology, because the structure can form hydrogen bonds. Use the same restrictive enzyme for plasmid and gene of interest. Dna ligase is used to form a bond between the vector, h bonds are not as strong. Insulin and growth hormone are the rst two human protein expressed via recombinant technique. Genomic dna libraries: it includes the whole genome cdna libraries: reverse transcription need to provide the sequence of the primer to allow the facility to make the primer for you. Agrose gel: many small pores in the gel. Band a, band b, band c are dna fragments.

Related Questions

1.Whati is Genetic Technology?What is bacterial transformation?
What is recombinant DNA technology?What is a vector?What is found in a typical plasmid?How can DNA sequenced use the dideoxy chain termination method? Describe the polymerase chain reaction (PCR). What are some advantages and limitations of this procedure?What is a cDNA? How are cDNAs and DNA microarrays used to study gene expression?Describe the natural function of restriction enzymes and explain how they are used in recombinant DNA technology,Explain how the creation of sticky ends by restriction enzymes is useful in producing a recombinant DNA moleculOutline the procedures for cloning a eukaryotic gene in a bacterial plasmid.Describe techniques that allow identification of recombinant cells that have taken up a gene of interest.How can you use the lacZ gene to facilitate cloning ?Distinguish between genomic and cDNA libraries.Define genomics and proteomics.Explain how gel electrophoresis is used to analyze nucleic acids and to distinguish between two alleles of a gene.How does dideoxy sequencing work? What is the significance of using dideoxy nucleotides?What are BACs, YACs, and contigs? What are some practical applications of DNA technology?What are the ways in which organisms can be genetically modified?

burgundylion987

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.)

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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

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Plasmids are put into bacterial cells by

restriction enzymes

DNA ligase

binding of cohesive sticky ends

transformation

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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

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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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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.

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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.

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Cloning allows for production of proteins in much larger amountsthan occurs in the cells from which the gene is isolated.

True

False

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Question 111 pts

Gene cloning is used to do all of the following except

Make insulin