Cloning and Sequencing Practice Flashcards

A comprehensive set of vocabulary flashcards covering nucleic acid extraction, agarose gel electrophoresis, PCR techniques, cloning methods, cancer genetics, and CRISPR-Cas9 technology based on the lab lecture notes.

Lysis

The process of breaking open cells to release their contents, typically achieved using a buffer with high salt and enzymes.

RNase A

An enzyme used during genomic DNA extraction to remove RNA from the lysate.

Proteinase K

An enzyme added to a sample to digest proteins and facilitate cell lysis and DNA purification.

Elution Buffer

A solution used to release DNA from the silica membrane in a chromatography column during the final stage of purification.

TAE Buffer

Tris-Acetate-EDTA; an electrophoresis buffer that provides better resolution for DNA fragments larger than $4\,kb$.

TBE Buffer

Tris-Borate-EDTA; an electrophoresis buffer that provides better resolution for $0.1$ to $3\,kb$ fragments and is used for voltages over $150\,V$.

SYBR Green

A fluorescent dye that binds to double-stranded DNA, used for visualization in agarose gels and quantification in real-time PCR.

DNA Ladder

Also known as a molecular weight marker, these are prepared DNA fragments of known sizes used to estimate the size of unknown DNA samples on a gel.

Bromophenol Blue

A tracking dye in gel loading buffer that typically appears as a dark blue, lower band during electrophoresis.

Xylene Cyanol FF

A tracking dye in gel loading buffer that typically appears as a light blue, upper band during electrophoresis.

PCR

Polymerase Chain Reaction; a technique for rapidly creating multiple copies of a DNA segment utilizing repeated cycles of DNA synthesis.

Kary Mullis

The biochemist who developed the polymerase chain reaction technique in 1983 and later won a Nobel Prize.

Thermal Cycler

An instrument that automates the PCR process by rapidly heating and cooling the reaction block to the temperatures required for each step.

Denaturation

The first step of the PCR cycle where double-stranded DNA is heated to $94^{\circ}C$ to separate it into single strands.

Annealing

The second step of the PCR cycle where the thermal cycler cools to $40\text{--}60^{\circ}C$ to allow primers to bind to the template strands.

Extension

The final step of the PCR cycle, occurring at $72^{\circ}C$, where DNA polymerase synthesizes new DNA strands starting from the primers.

Taq DNA Polymerase

A heat-stable enzyme that synthesizes new DNA strands by linking free nucleotides in an order determined by the template strand.

dNTPs

Deoxynucleoside triphosphates; the four individual bases (dATP, dTTP, dCTP, dGTP) that serve as building blocks for the new DNA polymer.

Melting Temperature (Tm)

The temperature at which half of the primers dissociate from the target DNA sequence.

Primer Dimers

An unwanted PCR artifact produced when primers hybridize at their $3'$-ends and act as a template for DNA polymerase.

GC-Clamp

The presence of one to three G or C bases at the $3'$-end of a primer to ensure strong and correct binding to the template.

Degenerate Primers

A mixture of primers with one or more substituted bases used to amplify target DNA when the exact sequence is unknown but conserved regions are identified.

Consensus Sequence

A representative DNA sequence derived from aligning homologous genes from different species to find conserved and variable base positions.

Semiconservative Replication

The method of DNA replication where each double-stranded product contains one original strand and one newly synthesized strand.

TP53

Sometimes called the 'guardian of the genome', this gene codes for the p53 protein which regulates the cell cycle and functions as a tumor suppressor.

SNP

Single Nucleotide Polymorphism; variation that occurs in the DNA sequence at a single nucleotide position.

Synonymous SNP

A variation in the DNA sequence that does not change the amino acid sequence of the protein product due to degeneracy in the genetic code.

Missense Mutation

A non-synonymous SNP that alters the amino acid sequence of the resulting protein product.

Nonsense Mutation

A non-synonymous SNP that results in a premature STOP codon, causing the protein product to be truncated.

DNA Ligase

An enzyme, such as T4 DNA ligase, that catalyzes the formation of a phosphodiester bond between the $3'$-hydroxyl and $5'$-phosphate ends of DNA.

Sticky Ends

DNA fragments with unpaired bases at the ends (overhangs) that are generated by certain restriction enzymes or Taq DNA polymerase.

Blunt Ends

DNA fragments where both strands are the same length with no overhangs; required for ligation into vectors like pJET1.2/blunt.

Competent Cells

Bacterial cells that have been treated, often with calcium chloride or electrical shocks, to enable them to take up foreign DNA.

Transformation

The process of introducing a plasmid or other foreign DNA into living bacterial cells so that it can be replicated.

pJET1.2/blunt Vector

A high copy number linearized plasmid designed for blunt-end cloning that allows for positive selection of transformants.

eco47IR

A lethal gene in the pJET1.2 vector that, when disrupted by a DNA insert, allows the transformed bacteria to survive on selective media.

Inoculation

The process of placing cells from an isolated bacterial colony into liquid nutrient medium to start a culture.

Reverse Transcriptase

An RNA-directed DNA polymerase that creates a DNA copy from a single-stranded mRNA template.

cDNA

Complementary DNA; the double-stranded DNA product synthesized during reverse transcription from an mRNA template.

QRT-PCR

Quantitative Real-Time PCR; an assay used to measure the specific amount of target DNA or RNA as amplification progresses using fluorescence.

Ct Value

Threshold cycle; the cycle number at which the fluorescence intensity of a sample crosses an arbitrary threshold in real-time PCR.

Cas9

A bacterial endonuclease that forms a double-strand break in DNA at a specific site determined by a guide RNA.

sgRNA

Single guide RNA; an engineered fusion of two regions (guiding and scaffold) that forms a complex with Cas9 to target specific DNA sites.

PAM

Protospacer Adjacent Motif; a sequence (specifically $5'-NGG$ for Cas9) required for the Cas9 enzyme to bind and cut the adjacent protospacer sequence.

HDR

Homology Directed Repair; a DNA repair mechanism in which enzymes patch a break using donor template DNA flanked by homology arms.