Recombinant DNA Technology and DNA Cloning

Comprehensive practice flashcards covering Recombinant DNA technology, DNA cloning vectors, library creation, PCR, sequencing methods, and blotting techniques.

A(n) __________ is defined as a genetically identical copy of a cell or organism.

Clone

Gene cloning was made possible by the discovery of __________ and plasmid DNA vectors.

Restriction Enzymes

Restriction enzymes primarily found in bacteria cut DNA by cleaving the __________ bond between adjacent nucleotides.

phosphodiester

A restriction site is a(n) __________, meaning the sequence of bases reads the same forward and backward on opposite strands.

palindrome

Bacteria prevent restriction enzymes from digesting their own DNA by using __________ DNA.

Methylated

Restriction enzymes that create overhanging single-stranded ends produce fragments with __________ ends.

Sticky

Ligating fragments with __________ ends results in a significantly lower yield and the potential for insertion in the opposite orientation.

blunt

The enzyme __________ is used to form a covalent bond between the sugar-phosphate residue of adjacent nucleotides to join DNA molecules.

DNA ligase

In agarose gel electrophoresis, a(n) __________ percentage gel is used to resolve smaller size fragments.

$$2\%$$

DNA is always negatively charged and migrates toward the __________ pole during electrophoresis.

positive

__________ is a staining dye that intercalates between DNA base pairs and fluoresces under ultraviolet light.

Ethidium bromide

Plasmid DNA is considered __________ DNA because it is found in the bacterial cytoplasm separate from the chromosome.

extrachromosomal

Calcium Chloride Transformation involves heat shocking a cell and DNA mixture for $30\,\text{sec}$ at __________.

$$42^{\circ}C$$

The technique of __________ applies a brief pulse of high voltage electricity to create tiny holes in the bacterial cell wall.

Electroporation

In blue-white selection, cells containing a recombinant plasmid appear as __________ colonies because the lacZ gene is non-functional.

white

A(n) __________ library contains DNA fragments representing the entire genome, including both introns and exons.

Genomic DNA

To create a cDNA library, mRNA is extracted and converted into double-stranded DNA using the enzyme __________.

reverse transcriptase

In colony hybridization, the filter is treated with a(n) __________ solution to lyse the cells and denature the DNA.

alkaline

Polymerase Chain Reaction (PCR) was developed in 1983 by __________.

Kary Mullis

The three stages of a PCR cycle are denaturation, __________, and extension.

Annealing

During the extension stage of PCR, DNA polymerase copies the target DNA at a temperature of __________ to __________.

$70^{\circ}C$; $75^{\circ}C$

The number of copies of target DNA after $N$ cycles of PCR is calculated using the equation __________.

$$2^N$$

Taq DNA polymerase was isolated from the species __________, which thrives in hot springs.

Thermus aquaticus

Taq polymerase adds a single __________ nucleotide to the $3'$ end of PCR products, which is utilized for cloning into T vectors.

adenine

The Sanger method of DNA sequencing is also known as __________ sequencing because it uses ddNTPs to halt synthesis.

chain termination

In automated sequencing, the light emitted by fluorescently labeled ddNTPs is converted into a nucleotide sequence called a(n) __________.

electropherogram

The Ion Torrent PGM system conducts sequencing by detecting the release of __________ on a semiconductor chip.

$$H^+$$

__________ blotting is a technique used to determine gene copy number or confirm PCR products by analyzing DNA on a filter.

Southern

__________ blot analysis is used to study gene expression by analyzing mRNA produced by a tissue.

Northern

In qPCR, __________ dye emits fluorescent light only when it binds to double-stranded DNA.

SYBR green

The technique used to identify the chromosomal location of a gene of interest is called __________.

Fluorescence in situ hybridization (FISH)