Biotechnology Lecture: PCR, Cloning, and GMOs

Flashcards covering biotechnology concepts including PCR, restriction enzymes, cloning vectors, and genetic engineering examples based on lecture notes.

Samuel E. Kelly Ethnic Cultural Center

The largest stand-alone multicultural center on a college campus in the nation, which serves as an inclusive space for historically marginalized students and hosts over 160 Registered Student Organizations.

DNA polymerase III

An enzyme involved in DNA replication that is responsible for replicating all DNA at the active sites.

DNA Polymerase I

An enzyme involved in the DNA replication process along with primase, ligase, and helicase.

DNA Denaturation

The process of separating double-stranded DNA into single strands by heating.

PCR (Polymerase Chain Reaction) ingredients

A combination of Source DNA, ATP, GTP, TTP, CTP, Polymerase, Buffer, and Primer Pairs.

PCR Cycle Steps

The repeating three-step process consisting of 1) Heat, 2) Cool, and 3) Extend.

PCR Primer Competition

Primers successfully bind to denatured DNA instead of the original strands reannealing because they are smaller (faster) and present in vastly higher concentrations.

PCR Amplification Rate

The exponential growth of DNA copies described by the formula $~2^{N+1}$ copies for $N$ cycles.

PCR 35th Cycle Yield

The approximate number of DNA copies produced after 35 cycles of PCR, totaling $68\text{ billion}$ copies.

Short Tandem Repeats (STR)

Highly variable sequences that exist at approximately $700,000$ unique locations (loci) throughout the human genome, used in forensics and paternity testing.

Restriction Enzymes

Bacterial enzymes that selectively degrade foreign DNA at specific sequences known as restriction sites, acting as a form of bacterial immunity.

Modification Enzyme

An enzyme that protects bacterial DNA by selectively methylating the restriction sites in the organism's own genome.

Type II Restriction Enzymes

The most commonly used subclass of restriction enzymes, which are typically homodimers with palindromic recognition sites of $4\text{-}8\text{ nt}$ in length.

Palindromic DNA Sequence

A sequence made of nucleic acids within a double helix that reads the same from $5'$ to $3'$ on one strand as it does from $5'$ to $3'$ on the complementary strand.

Sticky Ends

DNA ends with overhanging single-stranded sequences produced by enzymes like $EcoR1$ that cut within a recognition site.

Vector

A DNA molecule, based on naturally occurring DNA, into which foreign DNA can be inserted and replicated within a host cell.

DNA Ligase (in cloning)

The enzyme used to join or ligate the cut vector and the DNA of interest to create Recombinant DNA.

Transformation

The step in cloning where the recombinant vector is introduced into a viable cell that will replicate it.

Plasmid Vector Capacity

A type of vector replicated in bacteria, such as $E\text{-}coli$, that can hold foreign DNA up to $20\text{ kb}$.

Bacterial Artificial Chromosomes (BACs)

Vectors utilized for large inserts, typically holding between $100\text{-}200\text{ kb}$ of foreign DNA, with a maximum capacity of $300\text{ kb}$.

Yeast Artificial Chromosomes (YACs)

Vectors used for very large genomic sequences, holding between $200\text{ kb}$ and $1\text{ MBp}$, with a maximum capacity of $5\text{ Mbp}$.

Ampicillin Selection

A method to eliminate non-transformed cells using a medium where only cells that took up the vector become antibiotic resistant.

X-gal Selection

A process used to determine if a vector contains an insert by identifying and ignoring cells that contain only empty vector sequences.

Multiple Cloning Site

A feature in an ideal vector that provides versatility by offering various sites where a fragment of interest can be inserted.

1980 Supreme Court Patent Ruling

A landmark decision where the U.S. Supreme Court approved the patenting of life specifically for a bacteria that eats oil.

Humulin

The first human insulin drug produced via recombinant DNA, approved for the market in $1982$.

Genetically Engineered (GE)

A specific term for an organism that contains a protein from a completely different species, a result that cannot be achieved through selective breeding.

Agrobacterium tumefaciens

A soil bacterium known as nature's genetic engineer that uses a pilus and a type IV secretion system to inject $T\text{-}DNA$ into plant cells.

Ti Plasmid

The tumor-inducing plasmid found in $A. tumefaciens$; it is modified for genetic engineering by replacing opine-synthesizing genes with desirable genes for transfer.

Roundup Ready Plants

Crops engineered with a modified version of the enzyme $ESPS\text{ synthase}$ that is resistant to the broad-spectrum herbicide $glyphosphate$.