Human Molecular Genetics - Recombinant DNA Technology

Flashcards covering recombinant DNA technology, cloning vectors, restriction enzymes, DNA libraries, and sequencing methods from the Human Molecular Genetics lecture.

Cloning Vectors

Molecules that hold large fragments of DNA and act to increase the number of copies through replication.

Plasmid

A type of cloning vector that grows in bacteria and carries only small pieces of DNA.

Bacteriophage

A cloning vector that grows in bacteria and destroys them.

Yeast Artificial Chromosome (YAC)

A cloning vector used for taking very large DNA inserts.

Expression Vectors

Vectors engineered with inducible promoters and special features to control the expression and purification of protein from a cloned gene.

Inducible Promoter

A promoter that is not active until an inducer molecule is provided to the growing cells, such as the Lac promoter induced by IPTG.

pUC19

A specific plasmid cloning vector that includes an origin of replication (ori), an $amp^R$ selectable marker, and a multiple cloning site.

Multiple Cloning Site (MCS)

Also known as a polylinker, this is a region in a vector containing multiple different restriction palindromes for DNA insertion.

Blue-white Color Selection

A screening test where the insertion of a DNA fragment disrupts the $lacZ^+$ gene, resulting in nonfunctional $\beta$-galactosidase and allowing selection of vectors with inserts.

Restriction Enzymes

Molecular scissors found in prokaryotes that cut double-stranded DNA at specific recognition sequences.

Palindrome

A DNA sequence that reads the same from left to right (5'-to-3') on the top strand as it does from right to left (5'-to-3') on the bottom strand.

SmaI

A restriction enzyme that cuts the sequence 5'-CCCGGG-3' to produce blunt ends.

BamHI

A restriction enzyme that cuts the sequence 5'-GGATCC-3' to produce 5' overhanging sticky ends.

PstI

A restriction enzyme that cuts the sequence 5'-CTGCAG-3' to produce 3' overhanging sticky ends.

DNA Ligase

An enzyme used to seal gaps in the DNA backbone by joining fragments together after they have annealed.

Genomic Library

A collection of DNA fragments representing the entire genome of an organism, including exons, introns, and promoters.

cDNA Library

A collection of double-stranded DNA synthesized from polyadenylated mRNA using reverse transcriptase, RNase H, DNA polymerase I, and DNA ligase.

Colony Hybridisation

A library screening technique that uses DNA probes and radioactivity or antibodies to identify genes of interest.

Southern Blot

A DNA analysis process used to verify that a section of DNA has been inserted into a genome; it relies on DNA digestion, electrophoresis, and hybridisation.

Protein-Protein Interactions (PPI)

Physical contacts between two or more proteins that are characterized as either stable or transient.

Yeast 2-hybrid

A technique based on the reconstitution of a functional transcription factor when two proteins of interest (bait and prey) interact within genetically modified yeast.

Single Nucleotide Polymorphisms (SNPs)

Single base-pair variations that account for approximately 90% of human genome variation, occurring about once every 100-300 bases.

Sanger Dideoxy Method

A DNA sequencing technique that uses dideoxynucleotides to stop chain elongation during PCR.

Dideoxynucleotide (ddNTP)

A DNA building block that lacks the normal OH group at the 3' position, preventing the addition of further nucleotides.