Recombinant DNA Technology Lecture Notes

Flashcards covering key terms and concepts related to recombinant DNA technology, cloning, PCR, and their applications.

Recombinant DNA Technology

The process of combining DNA from different sources using molecular biology techniques.

Plasmid

A small, circular, extrachromosomal DNA molecule in bacteria, often used as a vector in genetic engineering.

Vector

A DNA molecule (like a plasmid) used to carry foreign genetic material into another cell where it can be replicated and/or expressed.

Restriction Enzyme

An enzyme that recognizes and cuts DNA at specific nucleotide sequences, often producing cohesive (sticky) ends.

Multiple Cloning Site (MCS)

A short DNA segment in a plasmid containing several restriction enzyme recognition sites, facilitating the insertion of foreign DNA.

Origin of Replication (Ori)

A specific DNA sequence where DNA replication initiates, ensuring the plasmid can be copied within a host cell.

lacZ gene

A reporter gene in plasmids, encoding beta-galactosidase, used in blue-white screening to identify recombinant colonies.

Ampicillin Resistance Gene (amp gene)

A gene on a plasmid that confers resistance to the antibiotic ampicillin, allowing for selection of transformed cells.

Transformation

The process by which bacterial cells take up foreign DNA from their environment.

Cohesive Ends (Sticky Ends)

Single-stranded overhangs created by certain restriction enzyme cuts, which can readily base-pair with complementary ends.

DNA Ligase

An enzyme that catalyzes the formation of phosphodiester bonds, covalently joining DNA fragments together.

Recombinant Plasmid

A plasmid into which a foreign DNA fragment, often containing a gene of interest, has been inserted.

Blue-White Screening

A method using the lacZ gene functionality to distinguish bacterial colonies containing recombinant plasmids (white) from non-recombinant plasmids (blue) on a selective medium.

Expression Vector

A type of plasmid designed to allow for the transcription and translation of a cloned foreign gene into protein within a host cell.

T7 Promoter

A strong promoter sequence often found in expression vectors (e.g., pET series), recognized by T7 RNA polymerase for efficient gene transcription.

His-Tag

A common affinity tag, typically a sequence of multiple histidine residues, genetically added to recombinant proteins to facilitate purification.

Taq DNA Polymerase

A heat-stable DNA polymerase enzyme isolated from Thermus aquaticus, commonly used in PCR due to its stability at high temperatures.

T Vector

A specialized cloning vector that contains single 3'-thymine (T) overhangs, designed for efficient ligation of PCR products with single 3'-adenine (A) overhangs.

Polymerase Chain Reaction (PCR)

A laboratory technique used to amplify specific DNA sequences exponentially, producing millions of copies from a small initial sample.

Primers (PCR)

Short, synthetic single-stranded DNA sequences that bind to complementary regions flanking the target DNA, initiating DNA synthesis in PCR.

Denaturation (PCR)

The first stage in a PCR cycle, involving heating the DNA to a high temperature to separate the double-stranded helix into single strands.

Annealing (PCR)

The second stage in a PCR cycle, where the temperature is lowered to allow primers to bind (anneal) to their complementary sequences on the single-stranded DNA templates.

Extension (PCR)

The third stage in a PCR cycle, where DNA polymerase synthesizes new DNA strands by adding nucleotides to the 3' end of the annealed primers.

PCR Applications

Diverse uses of PCR technology, including amplification of rare DNA, human genetic testing, forensic DNA analysis, DNA cloning, gene expression studies, paternity testing, human remains identification, and diagnostic tests for pathogens.