Molecular Biology and Genetic Engineering Methods

Flashcards covering key concepts and techniques discussed in the lecture notes.

What is the focus of molecular biology?

The mechanisms by which cellular processes are carried out by biological macromolecules, with emphasis on genes and genomes.

What two items are essential in a molecular biologist's toolkit?

Restriction endonucleases and cloning vectors

What is a restriction endonuclease?

An enzyme that recognizes specific short sequences of DNA and cleaves the duplex.

What is a cloning vector?

DNA (often derived from a plasmid or bacteriophage genome) that can be used to propagate an incorporated DNA sequence in a host cell. They contain selectable markers and replication origins.

What is the function of nucleases?

Enzymes that degrade nucleic acids by hydrolyzing the ester bond within a phosphodiester bond.

What is the function of phosphatases?

Hydrolyze the ester bond in a phosphomonoester bond.

What is an endonuclease?

A nuclease that cleaves phosphoester bonds within a nucleic acid chain, specific for RNA or single/double-stranded DNA.

What is an exonuclease?

A nuclease that cleaves phosphoester bonds one at a time from the end of a polynucleotide chain, specific for either the 5′ or 3′ end of DNA or RNA.

How can restriction endonucleases be used?

To cleave DNA into defined fragments.

What is a restriction map?

A linear sequence of sites separated by defined distances on DNA; generated by using the overlaps between fragments generated by different restriction enzymes.

What is recombinant DNA?

A DNA molecule created by joining together two or more molecules from different sources.

What does ligating (or ligation) mean?

The process of joining together two DNA fragments.

What does subclone mean?

The process of breaking a cloned fragment into smaller fragments for further cloning.

What is a multiple cloning site (MCS)?

A sequence of DNA containing a series of tandem restriction endonuclease sites used in cloning vectors for creating recombinant molecules.

What is transformation?

The acquisition of new genetic material by incorporation of added exogenous, nonviral DNA.

What does blue/white selection allow?

Identification of bacteria that contain the vector plasmid and vector plasmids that contain an insert.

Name four types of cloning vectors.

Bacterial plasmids, phages, cosmids, or yeast artificial chromosomes.

What is a shuttle vector?

A vector that can be propagated in more than one type of host cell.

What is an expression vector?

A vector that contain promoters that allow transcription of any cloned gene.

What are reporter genes?

They can be used to measure promoter activity or tissue-specific expression.

What is a probe?

A radioactive nucleic acid, DNA or RNA, used to identify a complementary fragment.

What is autoradiography?

A method of capturing an image of radioactive materials on film.

What is in situ hybridization?

Hybridization of a probe to intact tissue to locate its complementary strand by autoradiography.

What does gel electrophoresis do?

Separates DNA fragments by size, using an electric current to cause the DNA to migrate toward a positive charge.

What does gradient centrifugation do?

Separates samples based on their density.

What do classical chain termination sequencing use?

dideoxynucleotides (ddNTPs) to terminate DNA synthesis at particular nucleotides.

What is a primer?

A single stranded nucleic acid molecule with a 3′ –OH used to initiate DNA polymerase replication of a paired template strand.

What is a key feature of DideoxyNTP sequencing?

It uses fluorescent tags.

What allows automated, high-throughput DNA sequencing?

Fluorescently tagged ddNTPs and capillary gel electrophoresis