9.1

What is DNA cloning?

Making many identical copies of a chosen DNA segment.

Why do scientists clone genes?

To study DNA, alter genes, or produce the proteins they encode.

What is recombinant DNA?

DNA made by covalently joining segments from two or more sources.

What is recombinant DNA technology?

Methods for cutting, joining, and propagating DNA from different sources.

What are the five main steps of DNA cloning?

Obtain DNA; choose vector; ligate DNA; introduce into host; select correct cells.

How can a gene of interest be obtained?

By restriction digestion, PCR amplification, or DNA synthesis.

What is a restriction endonuclease?

An enzyme that cuts DNA at specific recognition sequences.

Why are type II restriction enzymes useful?

They cut within defined recognition sites and generally need no ATP.

What is a palindromic DNA recognition sequence?

A sequence that reads the same 5' to 3' on both complementary strands.

What is the recognition site of EcoRI?

5'-GAATTC-3'.

What are sticky ends?

DNA ends with short single-stranded overhangs that can base-pair.

What are blunt ends?

DNA ends cut straight across, with no single-stranded overhang.

Why are complementary sticky ends useful?

They base-pair and make DNA fragments easier to ligate.

Can blunt DNA ends be ligated?

Yes, but generally less efficiently than compatible sticky ends.

What does DNA ligase do?

Forms phosphodiester bonds to join DNA fragments.

What does DNA ligase require in this cloning example?

ATP or a similar energy-providing cofactor.

Why cut a vector and insert with the same restriction enzyme?

To create compatible DNA ends for ligation.

What is a DNA linker?

A synthetic DNA fragment used to bridge or add useful sequences to DNA.

What is a multiple cloning site (MCS)?

A short DNA region containing several unique restriction sites.

How can PCR primers help with cloning?

They can add restriction sites to the ends of amplified DNA.

What is a cloning vector?

A DNA carrier that can replicate with an inserted DNA fragment in a host.

What is a plasmid?

A small, usually circular DNA molecule that replicates separately from a chromosome.

What is an origin of replication (ori)?

A DNA sequence where replication begins.

What is pBR322?

An E. coli cloning plasmid with an ori and ampicillin/tetracycline resistance genes.

How large is pBR322?

4,361 base pairs.

What are AmpR and TetR in pBR322?

Genes conferring resistance to ampicillin and tetracycline.

What happens if foreign DNA disrupts pBR322's AmpR gene?

Cells may resist tetracycline but become sensitive to ampicillin.

What is a selectable marker?

A gene that lets cells survive or die under specified selection conditions.

What is a screenable marker?

A gene producing an observable signal, such as color or fluorescence.

What is bacterial transformation?

Introduction of foreign DNA, such as a plasmid, into bacterial cells.

How can heat shock help transformation?

A temperature change helps some treated bacteria take up plasmid DNA.

What is electroporation?

An electric pulse briefly makes cell membranes more permeable to DNA.

Why are plasmid cloning vectors limited for large DNA?

Transformation and stable cloning become harder as insert size increases.

What is a BAC?

A bacterial artificial chromosome used to clone very large DNA fragments.

What insert sizes can BACs commonly carry?

About 100,000 to 300,000 base pairs.

Why are BACs maintained at low copy number?

It helps reduce unwanted recombination of large inserted DNA.

What do par genes do in BACs?

Help distribute the BAC reliably to daughter cells during division.

Which antibiotic marker is described for the BAC vector?

Chloramphenicol resistance (CamR).

How does blue-white screening work in the BAC example?

Intact lacZ makes blue colonies; disrupted lacZ makes white colonies.

What does a white colony suggest in BAC blue-white screening?

A DNA insert may have disrupted the lacZ gene.

What is a YAC?

A yeast artificial chromosome used to clone very large DNA segments.

What essential features does a YAC need?

Yeast ori, centromere, telomeres, and selectable markers.

What does the centromere do in a YAC?

Helps the chromosome segregate correctly during cell division.

What do telomeres do in a YAC?

Protect and stabilize the ends of a linear chromosome.

What happens to a YAC lacking a telomere?

It is rapidly degraded.

What is a shuttle vector?

A vector that can be propagated in two or more host species.

What is the difference between cloning and expression vectors?

Cloning vectors copy DNA; expression vectors enable protein production.

What is an expression vector?

A vector with regulatory sequences needed to express an inserted gene.

What does a promoter do?

Directs where transcription of a gene begins.

What does a bacterial ribosome-binding site do?

Helps ribosomes initiate translation of the mRNA.

Why might a eukaryotic gene need bacterial control sequences?

Its native regulatory signals may not work in bacteria.

Why is E. coli commonly used to express proteins?

It is inexpensive, grows quickly, and is easy to manipulate.

What is a limitation of expressing eukaryotic proteins in bacteria?

They may misfold or lack necessary posttranslational modifications.

What are inclusion bodies?

Insoluble aggregates of improperly folded proteins in cells.

What is a drawback of the bacterial lac expression system?

It can be leaky, allowing expression even without inducer.

How does a T7 expression system work?

T7 RNA polymerase transcribes a gene placed under a T7 promoter.

What is one advantage of yeast for protein expression?

As a eukaryote, it can better fold and modify some eukaryotic proteins.

How do GAL1/GAL10 promoters respond to sugars?

Galactose induces expression; glucose represses it.

What are baculoviruses used for in biotechnology?

Delivering genes for protein expression in insect cells.

What is a bacmid?

A large DNA vector containing a baculovirus genome that can be maintained in bacteria.

How are cloned genes often delivered into mammalian cells?

With engineered viral vectors, including adenoviruses and retroviruses.

Transient vs. permanent expression in mammalian cells?

Transient DNA is not stably maintained; integrated DNA may persist long term.

What is an advantage and disadvantage of mammalian expression?

Suitable protein processing; high culture cost.

What is site-directed mutagenesis?

Deliberately changing a specific DNA sequence to alter a protein.

Why use site-directed mutagenesis?

To test how chosen amino acids affect protein structure or function.

How does oligonucleotide-directed mutagenesis work?

Primers with intended mismatches direct DNA synthesis containing the mutation.

What change was made to RecA in the chapter?

Lysine at position 72 was changed to arginine (Lys72Arg).

What happened to the RecA Lys72Arg variant?

It could bind ATP but could not hydrolyze ATP effectively.

What did the RecA experiment reveal?

Lys72 is important for ATP hydrolysis, not just ATP binding.

How can researchers remove a protein domain?

Delete the DNA segment encoding that domain from the cloned gene.

What is a fusion protein?

A protein made by joining coding sequences from different proteins or parts.

Why attach a purification tag to a protein?

To provide a binding handle for affinity purification.

What is affinity chromatography?

Purification based on specific binding to a ligand attached to a column.

What does a GST tag bind?

Glutathione.

What does a His6 tag bind?

Immobilized metal ions such as Ni2+.

What does a protein A tag bind?

The Fc portion of immunoglobulin G (IgG).

Can a protein purification tag be removed?

Yes; a protease can cleave a designed site between tag and protein.

What is a potential problem with protein tags?

They may change protein folding, stability, or activity.

What is reverse transcription PCR (RT-PCR)?

RNA is converted to cDNA, then amplified using PCR.

What enzyme converts RNA into cDNA?

Reverse transcriptase.

What is quantitative PCR (qPCR)?

PCR that measures DNA amplification using fluorescence in real time.

What does an earlier qPCR threshold cycle suggest?

More starting target nucleic acid, assuming comparable amplification efficiency.

What can RT-qPCR measure?

Relative abundance of a specific RNA after reverse transcription.

What is a DNA library?

A collection of many different cloned DNA fragments.

What is a cDNA library?

A library of DNA copies made from RNA expressed in sampled cells.

What does a gene's presence in a cDNA library indicate?

The gene was expressed in the cells under the sampled conditions.

Why do cDNA libraries generally lack introns?

They are made from mature mRNA after RNA splicing.

What is a combinatorial gene library?

A collection of many sequence variants of a particular gene.

Why screen a combinatorial gene library?

To find gene variants that change or improve protein properties.

How do bacteria protect their DNA from their own restriction enzymes?

Methylation of recognition sites by corresponding DNA methylases.

What is a restriction-modification system?

A restriction enzyme paired with a methylase that protects host DNA.

What does DNA polymerase I do in recombinant DNA methods?

Fills gaps in DNA duplexes by adding nucleotides to 3' ends.

What does polynucleotide kinase do?

Adds a phosphate to a polynucleotide's 5'-OH end.

What does alkaline phosphatase do?

Removes terminal phosphate groups from DNA ends.

What does terminal transferase do?

Adds nucleotide tails to the 3'-OH ends of DNA.

What does exonuclease III do?

Removes nucleotides from the 3' ends of double-stranded DNA.

What does bacteriophage lambda exonuclease do?

Removes nucleotides from the 5' ends of duplex DNA.