Molecular Biology Techniques: Nucleases, Cloning, Sequencing, and Genetic Engineering

What are nucleases and how are they used?

Nucleases hydrolyze phosphodiester bonds in nucleic acids; endonucleases cut within a chain and exonucleases remove nucleotides from an end.

What is the purpose of DNA cloning?

DNA cloning copies a DNA fragment by inserting it into a vector that can replicate in a host cell.

What are the steps of DNA cloning?

Cut insert and vector with compatible restriction enzymes, ligate them to form recombinant DNA, introduce into cells by transformation, then select clones carrying the insert.

What are common cloning vectors?

Plasmids: small circular DNA for routine cloning; Phage vectors: for larger inserts; Cosmids: hybrid vectors for larger DNA; YACs: yeast artificial chromosomes for very large fragments.

What are specialized vectors in cloning?

Shuttle vectors work in multiple hosts, expression vectors drive transcription, and reporter vectors measure gene or promoter activity.

How are nucleic acids detected?

A labeled probe hybridizes to a complementary nucleic acid sequence.

What are methods for detecting nucleic acids?

Autoradiography for radioactive probes and in situ hybridization/FISH for locating sequences in intact cells or tissues.

What is gel electrophoresis used for?

Gel electrophoresis separates DNA fragments by size as they migrate toward the positive electrode.

How is DNA sequenced?

Classical sequencing uses dideoxynucleotides (ddNTPs) to terminate synthesis; automated sequencing uses fluorescent ddNTPs and capillary electrophoresis.

What is PCR?

PCR exponentially amplifies a target DNA sequence using primers and a thermostable DNA polymerase through repeated cycles.

What is RT-PCR?

RT-PCR uses reverse transcriptase to convert RNA into DNA, then amplifies it by PCR to study RNA expression.

What is real-time PCR?

Real-time PCR measures amplification as it happens and is more sensitive and quantitative.

What is a Southern blot?

Southern blot detects specific DNA sequences after transfer from a gel to a membrane using a labeled probe.

What is a Northern blot?

Northern blot is similar to Southern blot but for detecting RNA sequences.

What is a Western blot?

Western blot separates proteins by SDS-PAGE, transfers them to a membrane, and detects them with antibodies.

What are DNA microarrays?

DNA microarrays contain many immobilized probes and detect gene expression or sequence presence by hybridization.

What is Chromatin Immunoprecipitation (ChIP)?

ChIP uses an antibody to pull down DNA bound to a specific protein and identifies the associated DNA.

What is genetic engineering?

Genetic engineering introduces, modifies, or replaces genes using recombinant DNA methods.

What are transgenics?

Transgenics are organisms made by introducing foreign DNA into the germline so the new DNA can be inherited.

How are transgenes introduced in mice?

Transgenes can be introduced into ES cells, used to make chimeras, and passed through the germline.

What does a low nanodrop number indicate?

Low nanodrop numbers indicate protein contamination.