Genetic Technologies II

Announcements:

• Homework for Lecture 40 (Wednesday) will consist of 6 pre-lecture questions instead of the usual 3 pre + 3 post questions.

• The deadline for checking the accuracy of grades entered into the ATLAS web gradebook is Wednesday.

• Wednesday is also the deadline for requesting a Conflict Final.

• Regular student hours will be held on Wednesday. Extra review sessions are scheduled for Friday and Monday from 11:30 AM to 1:00 PM in 124 Burrill Hall.

• The Final Exam is on Monday, May 12, from 7:00 PM to 10:00 PM in the same lecture hall.

• Utilize the Pearson AI Study Tool to create personalized problem sets and review material.

Gene Cloning Using Vectors

• Requirements of plasmid vector molecules:

  • Must have an origin of replication (ori).

  • Must have a restriction site for the enzyme used in cloning.

  • Ideally, it should have restriction sites for many restriction enzymes.

  • Must have a method to select for the vector in the cell.

  • Should have a way to distinguish between the vector alone and the vector with the insert.

Typical Plasmid Cloning Vector

• Components:

  • Antibiotic Resistance Gene (e.g., ampicillin resistance)

  • Reporter Gene (e.g., lacZ gene)

  • Polylinker

  • ori

• Polylinker:

  • Has an origin of replication.

  • Has restriction sites in the polylinker.

  • Has a way to select for the vector.

  • Has a way to screen for the insert.

• Polylinker is a collection of unique restriction sites.

  • Located in the unconserved region of the promoter site for the reporter gene.

  • Insertion of DNA into the polylinker destroys the promoter, turning off the reporter gene.

• Example of a polylinker sequence:

  • 5'-gaattcgagctcggtacccggggatcctctagagtcgacctgcaggcatgcaagcttggc-3'

  • Restriction sites: EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, SalI, PstI, SphI, HindIII

Introducing Recombinant Vector into Host Cell

• The next step in the cloning process involves introducing the recombinant vector into a host cell.

• Bacteria like E. coli can be made transiently permeable to DNA in their surroundings, allowing the DNA to enter the cell – this process is called transformation.

• If the DNA is carried into the cell by a phage, the process is called transduction.

Replication of Recombinant Vector

• Host cell machinery replicates multiple copies of the recombinant vector, including the foreign DNA insert.

• The cloning process is now complete.

• Each cell that arises from the original host cell will also have multiple copies of the recombinant vector.

• These cells are called cell clones.

First Cloning Experiment

• Toad DNA was inserted into a bacterial plasmid vector to create a toad-bacterial DNA recombinant plasmid.

• This recombinant plasmid was then introduced into live bacteria via transformation.

• The recombinant bacteria expressed the toad DNA.

Why Clone?

• Amplification!

• Cloning allows us to obtain 10 times the amount of DNA for a typical gene of interest in 1 liter of E. coli culture compared to what we could obtain from every cell in an average adult human; cloning works to produce a large amount of foreign DNA in a host.

• Applications of cloned DNA:

  • Produce large amounts of protein from that gene.

  • Produce large amounts of the DNA itself to study.

    • Sequence, look for promoters, introns, coding regions, etc.

• Either way, we start with the construction of a library.

Preparation of Genomic Library

• Digest organism’s genome with restriction enzymes; depending on the genome size, this could result in hundreds or thousands of fragments.

• Digest the vector of choice with the same restriction enzymes.

• Ligate genome fragments with vector molecules; establish conditions where each vector takes in a single fragment.

• Transform living E. coli with potential recombinant vectors; try to allow only one vector molecule to enter a given host cell; each transformed cell carries a different piece of the genome.

• Collectively, these transformed cells are called a genomic library and represent the organism’s entire genome.

Genomic Library

• Overview of genomic library preparation:

  • Genomic DNA is cleaved with a restriction endonuclease.

  • The resulting fragments are inserted into a vector that has been cut with the same restriction enzyme as the genomic DNA.

  • The recombinant plasmids are introduced into an E. coli host strain, creating a library of genomic clones.

Experiment Success

• How do I know if my experiment was successful?

Genomic Library Limitations

• Why won’t a genomic library work for producing protein (e.g., insulin) from a eukaryotic gene?

  • Genomic libraries contain total chromosomal DNA, which means introns of eukaryotic genes are present.

  • Bacteria don’t know how to remove introns.

  • However, learning about retroviruses helped us overcome this obstacle.

    • Use reverse transcriptase to make DNA from the processed mRNA, then insert that DNA into a vector.

• Another limitation is that we need to know what cells are actively producing the protein made from the gene we want.

• Instead of a genomic library, produce a cDNA library.

Preparation of cDNA Library

• Harvest appropriate tissue; isolate processed mature mRNA.

• Use Reverse Transcriptase to make an RNA-DNA hybrid.

• Degrade the RNA, leaving only single-stranded DNA.

• Use DNA Polymerase to make a complementary strand, now you have double-stranded DNA (cDNA).

• Insert cDNA into a vector and transform into host cells.

• The gene can be transcribed and then translated by the host, without needing to remove introns.

• cDNAs do not include promoters or SD sequences; these come from the vector.

cDNA Library

• Total cell mRNA, representing genes being expressed in a particular cell type, is used.

• Reverse transcriptase and DNA polymerase are used to create double-stranded cDNAs.

• cDNAs are inserted into a vector.

• The vectors are introduced into E. coli host strain, creating a plasmid containing cDNA library of cDNA clones.

Genomic vs cDNA Library

• Important to remember that a genomic library represents the entire genome of the organism; each recombinant vector is a “piece of the puzzle”.

• A cDNA library, because it comes originally from processed mRNAs in a cell, represents only the proteins that were being made in the cells you harvested at the time you harvested them.

Gene Cloning Using PCR

Advantages and Limitations of PCR

• Advantages of PCR

  • Speed and relative ease-of-use

  • All in vitro, so fewer variables

• Limitations of PCR

  • Flanking sequences around target must be known

  • Contaminating DNA can be amplified