AS.020.151 Chapter Notes - Chapter 13: Gel Electrophoresis, Dna Ligase, Electroporation

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violetcat91

12 Jun 2018

School

JHU

Department

AS Biology

Course

AS.020.151

Professor

Shingles, Richard; Pearlman, Rebecca Shari; Roberson, Christov

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13.1 Recombinant DNA can be Made in the Laboratory

Conventional*breeding- many*generations,*only*one*species'*genes

Biotechnology- as*little*as*one*generation,*genes*from*any*organism

Recombinant+DNA- DNA*molecule*made*in*the*lab*derived*from*more*than*one*organism*(at*least*two*genetic*

sources)

Three+key+tools:

Restriction+enzymes for*cutting*DNA*into*fragments

•

Gel+electrophoresis for*analysis*and*purification

•

DNA+ligase for*binding*fragments*in*new*combinations

•

Restriction*enzymes

-Cut*DNA*at*specific*sequences*called*restriction*sites

-Cut*palindromic*sequences*(complementary*sequence*5'3*reads*same*as*original*5'3)

Bacteria*fight*invading*viruses- restriction*enzymes*recognize*foreign*DNA*and*cut*up*

Gel*electrophoresis

-DNA*fragments*separate,*indicating…

-number*of*fragments

-size*of*fragments

-relative*abundance*of*fragment*(intensity)

DNA*Ligase

-Enzyme*that*catalyzes*joining*fragments*of*DNA

-Some*with*different*DNA,*some*same

-So*some*recombinant*and*some*normal

13.2 DNA Can Genetically Transform Cells and Organisms

Recombinant*DNA*technology*can*be*used*to*clone genes

Transformation:*Recombinant*DNA*cloned*by*inserting*it*into*host*cell*(transfection if*host*is*animal)

Altered*host*is*transgenic

Methods for*inserting*the*recombinant*DNA:

-Chemicals*to*make*plasma*membrane*more*permeable

-Electroporation:*shock*creates*temporary*pores

-Viruses*and*bacteria*can*carry*recombinant*DNA*into*cells

-Inject*recombinant*DNA*into*nucleus*of*fertilized*egg->*transgenic*animals

-Gene*guns*can*shoot*host*cell*with*particles*of*DNA

Most*eukaryotic*genes*too*large*for*plasmid

Viruses*can*be*used*as*vectors

Viruses*infect*cells*naturally->*advantage*over*plasmids

Reporter+gene-gene*whose*presence*is*easily*observed

-like*gene*for*green*fluorescent*protein

13.3 Genes Come From Various Sources and Can be Manipulated

DNA*fragments*used*for*cloning*come*from*four*sources:

-gene*libraries

-reverse*transcription*from*mRNA

-products*of*PCR

-artificial*synthesis*or*mutation*of*DNA

Synthetic*DNA*can*be*manipulated*to*create*specific*mutations*to*study*consequences

Mutagenesis- revealed*many*cause*and*effect*relationships*(like*signal*sequences)

Knockout+experiment-inactivates*a*gene*so*that*it*is*not*transcribed*and*translated*into*a*protein

Another*way*to*study*gene*function:*block+translation

RNAi+(RNA+interference)- translation*of*mRNA*can*be*blocked*by*miRNAs*(degrade)*and*siRNAs*(bind*

and*degrade)

miRNA*and*siRNA- antisense+RNA

-can*be*synthesized*and*added*to*cells*to*prevent*translation*(effects*of*missing*protein*

determined)

13.4 Biotechnology Has Wide Applications

Almost*any*gene*can*be*inserted*into*bacteria/yeast*and*then*cells*induced*to*make*large*quantities

-Requires*specialized*expression+vectors (with*extra*genes*for*transgene*to*be*expressed*in*the*host*

cell)

Expression*vectors

Allow*to*produce*large*amounts*of*desired*protein

A*eukaryotic*expression*vector*has*a*promoter*and*poly-A*sequence

Pharming- production*of*pharmaceuticals*in*farm*plants*or*animals

Ie.*Transgenes*inserted*next*to*promoter*for*a*protein*in*milk

Transgenic*animal*produces*more*protein*in*its*milk

Bioremediation- use,*by*humans,*of*organisms*to*remove*contaminants*from*the*environment

Composting*and*wastewater*treatment

Concerns*of*biotechnology:

-Genetic*manipulation*is*unnaturally*interfering*in*nature

-Genetically*altered*foods*are*unsafe*to*eat

-Genetically*altered*crop*plants*are*dangerous*to*the*environment

Biotechnology*(Ch.*13)

Thursday, December 14, 2017

6:25 PM

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13.1 Recombinant DNA can be Made in the Laboratory

Conventional*breeding- many*generations,*only*one*species'*genes

Biotechnology- as*little*as*one*generation,*genes*from*any*organism

Recombinant+DNA- DNA*molecule*made*in*the*lab*derived*from*more*than*one*organism*(at*least*two*genetic*

sources)

Three+key+tools:

Restriction+enzymes for*cutting*DNA*into*fragments

•

Gel+electrophoresis for*analysis*and*purification

•

DNA+ligase for*binding*fragments*in*new*combinations

•

Restriction*enzymes

-Cut*DNA*at*specific*sequences*called*restriction*sites

-Cut*palindromic*sequences*(complementary*sequence*5'3*reads*same*as*original*5'3)

Bacteria*fight*invading*viruses- restriction*enzymes*recognize*foreign*DNA*and*cut*up*

Gel*electrophoresis

-DNA*fragments*separate,*indicating…

-number*of*fragments

-size*of*fragments

-relative*abundance*of*fragment*(intensity)

DNA*Ligase

-Enzyme*that*catalyzes*joining*fragments*of*DNA

-Some*with*different*DNA,*some*same

-So*some*recombinant*and*some*normal

13.2 DNA Can Genetically Transform Cells and Organisms

Recombinant*DNA*technology*can*be*used*to*clone genes

Transformation:*Recombinant*DNA*cloned*by*inserting*it*into*host*cell*(transfection if*host*is*animal)

Altered*host*is*transgenic

Methods for*inserting*the*recombinant*DNA:

-Chemicals*to*make*plasma*membrane*more*permeable

-Electroporation:*shock*creates*temporary*pores

-Viruses*and*bacteria*can*carry*recombinant*DNA*into*cells

-Inject*recombinant*DNA*into*nucleus*of*fertilized*egg->*transgenic*animals

-Gene*guns*can*shoot*host*cell*with*particles*of*DNA

Most*eukaryotic*genes*too*large*for*plasmid

Viruses*can*be*used*as*vectors

Viruses*infect*cells*naturally->*advantage*over*plasmids

Reporter+gene-gene*whose*presence*is*easily*observed

-like*gene*for*green*fluorescent*protein

13.3 Genes Come From Various Sources and Can be Manipulated

DNA*fragments*used*for*cloning*come*from*four*sources:

-gene*libraries

-reverse*transcription*from*mRNA

-products*of*PCR

-artificial*synthesis*or*mutation*of*DNA

Synthetic*DNA*can*be*manipulated*to*create*specific*mutations*to*study*consequences

Mutagenesis- revealed*many*cause*and*effect*relationships*(like*signal*sequences)

Knockout+experiment-inactivates*a*gene*so*that*it*is*not*transcribed*and*translated*into*a*protein

Another*way*to*study*gene*function:*block+translation

RNAi+(RNA+interference)- translation*of*mRNA*can*be*blocked*by*miRNAs*(degrade)*and*siRNAs*(bind*

and*degrade)

miRNA*and*siRNA- antisense+RNA

-can*be*synthesized*and*added*to*cells*to*prevent*translation*(effects*of*missing*protein*

determined)

13.4 Biotechnology Has Wide Applications

Almost*any*gene*can*be*inserted*into*bacteria/yeast*and*then*cells*induced*to*make*large*quantities

-Requires*specialized*expression+vectors (with*extra*genes*for*transgene*to*be*expressed*in*the*host*

cell)

Expression*vectors

Allow*to*produce*large*amounts*of*desired*protein

A*eukaryotic*expression*vector*has*a*promoter*and*poly-A*sequence

Pharming- production*of*pharmaceuticals*in*farm*plants*or*animals

Ie.*Transgenes*inserted*next*to*promoter*for*a*protein*in*milk

Transgenic*animal*produces*more*protein*in*its*milk

Bioremediation- use,*by*humans,*of*organisms*to*remove*contaminants*from*the*environment

Composting*and*wastewater*treatment

Concerns*of*biotechnology:

-Genetic*manipulation*is*unnaturally*interfering*in*nature

-Genetically*altered*foods*are*unsafe*to*eat

-Genetically*altered*crop*plants*are*dangerous*to*the*environment

Biotechnology*(Ch.*13)

Thursday, December 14, 2017

6:25 PM

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This preview shows pages 1-2 of the document.
Unlock all 5 pages and 3 million more documents.

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Document Summary

13. 1 recombinant dna can be made in the laboratory. Conventional breeding- many generations, only one species" genes. Biotechnology- as little as one generation, genes from any organism. Recombinant dna- dna molecule made in the lab derived from more than one organism (a sources) Dna ligase for binding fragments in new combinations. Cut dna at specific sequences called restriction sites. Cut palindromic sequences (complementary sequence 5"3 reads same as original 5"3) Bacteria fight invading viruses- restriction enzymes recognize foreign dna and cut up. 13. 2 dna can genetically transform cells and organisms. Recombinant dna technology can be used to clone genes. Transformation: recombinant dna cloned by inserting it into host cell (transfection if hos. Viruses and bacteria can carry recombinant dna into cells. Inject recombinant dna into nucleus of fertilized egg-> transgenic animals. Gene guns can shoot host cell with particles of dna. Viruses can be used as vectors ganism (at least two genetic ginal 5"3) if host is animal)

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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AS.020.151 Chapter Notes - Chapter 13: Gel Electrophoresis, Dna Ligase, Electroporation

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