Genetics Ch.20- Recombinant DNA Technology Review

These flashcards cover fundamental concepts, techniques, and tools relevant to recombinant DNA technology.

What are the two key tools that initiated recombinant DNA technology?

Restriction enzymes and DNA cloning vectors.

What is recombinant DNA?

Joining of DNA molecules produced by artificially joining DNA from different biological sources.

What is the role of restriction enzymes?

They cut DNA at specific recognition sequences (restriction sites) to produce restriction fragments.

What are restriction fragments?

DNA fragments produced when restriction enzymes cleave DNA.

What is a palindrome in genetics?

A symmetry exhibited by recognition sequences where the nucleotide sequence reads the same on both strands.

What are sticky ends?

Fragments produced by restriction enzymes that have overhangs.

What are blunt ends?

Fragments produced by restriction enzymes with double-stranded ends.

What is the function of DNA ligase?

It seals the phosphodiester backbone and joins restriction fragments covalently.

What are vectors in recombinant DNA technology?

Carrier DNA molecules that replicate cloned DNA fragments in host cells.

What are the characteristics of a vector?

Must replicate independently, have several restriction enzyme sites, and carry a selectable gene marker.

What is a plasmid?

An extrachromosomal double-stranded DNA molecule that replicates independently from chromosomes.

What is the primary use of plasmids in DNA cloning?

They are genetically modified to contain restriction sites and marker genes for selection.

How are plasmids introduced into bacteria?

Via transformation using calcium ions and heat shock or electroporation.

What is a multiple cloning site?

A region containing restriction sites for commonly used restriction enzymes for cloning different fragments.

What is blue-white screening?

A method to identify cells containing recombinant and nonrecombinant DNA using lacZ gene.

What is the purpose of the lacZ gene in blue-white screening?

It encodes galactosidase which cleaves X-gal to turn blue in nonrecombinant plasmid.

What are BACs and YACs used for?

They are vectors used to clone large fragments of DNA.

What is the function of expression vectors?

Designed to ensure mRNA expression of cloned genes to produce large quantities of proteins.

Why is Saccharomyces cerevisiae commonly used in DNA cloning?

It is easy to manipulate, has been extensively studied, and can posttranslationally modify eukaryotic proteins.

What are DNA libraries?

Collections of cloned DNA, which can be genomic or cDNA.

What is a genomic library?

A collection that contains at least one copy of all sequences in the genome of interest.

What does a cDNA library consist of?

Complementary DNA copies made from mRNAs present in the cell population.

How is cDNA synthesized?

By isolating mRNA and using reverse transcriptase to make DNA copies.

What is the role of a probe in library screening?

To screen the library and isolate specific genes of interest.

What is PCR (Polymerase Chain Reaction)?

A rapid method of DNA cloning that amplifies specific DNA sequences without using host cells.

What are the three steps of PCR?

Denaturation, primer annealing, and extension.

What limitation does PCR have?

Some sequence information is needed to synthesize primers, and it cannot amplify long DNA segments.

What is RT-PCR?

A methodology for studying gene expression by generating cDNA from mRNA.

What is the purpose of Southern blotting?

To identify which clones in a library contain a given DNA sequence.

What is a restriction map?

A diagram that shows the number, order, and distances of restriction enzyme cleavage sites on DNA.

What technique is used to visualize DNA fragments in gel electrophoresis?

Staining with ethidium bromide and illuminating with UV light.

What is the function of fluorescent in situ hybridization (FISH)?

To hybridize a probe directly to chromosomes or RNA without blotting.

What is the most common method of DNA sequencing?

Dideoxynucleotide chain-termination sequencing (Sanger sequencing).

What is the function of dideoxynucleotides in DNA sequencing?

They terminate DNA synthesis when incorporated into the growing DNA strand.

How did computer-automated DNA sequencing impact research?

It enabled rapid progress in large-scale projects like the Human Genome Project.

What is gene targeting?

Manipulating a specific allele or locus in the genome to learn its function.

What are knockout mice used for?

To disrupt specific genes to study the consequences and gene functions.

What is the Cre-lox system?

A method for creating conditional knockout animals, allowing targeted gene disruption at specific times.

What are transgenic animals?

Animals that contain a transgene, which can express or overexpress a particular gene.

What does CRISPR-Cas9 allow researchers to do?

It enables gene editing by creating precise changes in DNA sequences.

What is complementary DNA (cDNA)?

DNA synthesized from an mRNA template using reverse transcriptase.

What does the term 'transgene' refer to?

A gene that has been transferred from one organism to another.

What applications does PCR have in genetics?

Screening for mutations and diagnosing genetic disorders.

What is the purpose of multiple cloning sites in vectors?

To enable cloning of a range of different DNA fragments.

How does blue-white screening determine successful cloning?

Cells with recombinant plasmids appear white, while those with nonrecombinant plasmids appear blue.

What is the importance of DNA polymerase in PCR?

It synthesizes new DNA strands during the amplification process.

What does the Southern blot technique use to identify DNA fragments?

Labeled probes that hybridize to complementary sequences.