SCH2226 Human Molecular Genetics - Lecture 6: Recombinant DNA Technology

Flashcards covering cloning and expression vectors, restriction enzymes, DNA libraries, Southern blotting, Yeast 2 Hybrid systems, and SNP identification methods from Human Molecular Genetics Lecture 6.

Cloning Vector

A DNA molecule that holds large fragments of DNA and acts 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 type of cloning vector that grows in and destroys bacteria.

Yeast Artificial Chromosome (YAC)

A cloning vector capable of taking very large DNA inserts.

Expression Vectors

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

Inducible Promoter

A promoter, such as the $lac$ promoter from $E. coli$, that is not active until a specific inducer molecule like IPTG is provided.

$$pUC19$$

A plasmid cloning vector ($2.686\,kb$) containing an origin of replication ($ori$), an ampicillin resistance gene ($amp^R$), and a part of the $\beta$-galactosidase gene ($lacZ$).

Multiple Cloning Site (MCS)

Also known as a polylinker, a region containing multiple different restriction palindromes into which DNA fragments can be inserted.

Blue-white color selection test

A screening method used to select for vectors with or without inserts based on the breakdown of X-gal by functional $\beta$-galactosidase.

Restriction Enzymes

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

Palindrome

A DNA sequence that reads the same from $5'$-to-$3'$ on both the top and bottom strands, such as $5'-GAATTC-3'$.

Blunt ends

DNA ends produced by restriction enzymes like $SmaI$ that cut straight across the double helix.

Sticky ends

Overhanging DNA ends produced by restriction enzymes like $BamHI$ or $PstI$ that allow fragments to anneal via complementary base pairing.

DNA ligase

An enzyme used to seal the gaps between DNA fragments after they have annealed via sticky ends.

Genomic Library

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

cDNA Library

A library created by synthesizing double-stranded complementary DNA from poly-adenylated mRNA using reverse transcriptase.

Colony hybridization

A common library screening technique using short DNA probes, radioactivity, or antibodies to identify genes of interest.

Southern Blot

A DNA analysis process developed by Edwin Southern in 1975 to verify the insertion of specific DNA sections into a genome using hybridization.

Protein-Protein Interactions (PPI)

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

Yeast 2 Hybrid (Y2H)

A system based on the reconstitution of a functional transcription factor (TF) to detect protein interactions in genetically modified yeast strands.

DNA Binding Domain (DBD)

One part of a transcription factor used in Yeast 2 Hybrid, often from the yeast TF $Gal4$ or the bacterial protein $LexA$.

Activation Domain (AD)

The second part of a transcription factor in Yeast 2 Hybrid that, when brought near the DBD by interacting proteins, activates a reporter gene.

Reporter Gene

A gene such as $HIS3$ or $LacZ$ used in Yeast 2 Hybrid to produce a specific phenotype (like growth or color change) when proteins interact.

Single Nucleotide Polymorphisms (SNPs)

Single base-pair variations that account for $90\%$ of human genome variation and occur about once in every $100$-$300$ bases.

Sanger dideoxy method

A DNA sequencing technique that relies on dideoxynucleotides to stop PCR chain elongation at specific bases.

Dideoxynucleoside triphosphate (ddNTP)

A DNA precursor that lacks the $OH$ group at the $3'$ position, preventing further chain elongation when incorporated into DNA.

Automated DNA sequence analysis

A method using fluorescent terminators and lasers where different colors represent the four bases: A (green), G (black), C (blue), and T (red).