SCH1111 Fundamental Biomedical Techniques: PCR & Other Molecular Techniques

A set of vocabulary flashcards covering basic molecular biology techniques, gel electrophoresis, restriction enzymes, PCR, and DNA sequencing methods based on lecture material.

Helicase

An enzyme that unwinds a portion of the parent DNA at the origin to form a replication fork during DNA replication.

DNA Polymerase III

An enzyme that binds to template strands and moves in a 3’ to 5’ direction, reading the sequence and replicating the new strand in a 5’ to 3’ direction.

Okazaki fragments

Discontinuous segments of DNA synthesized on the lagging strand during DNA replication.

DNA Ligase

An enzyme that joins or ligates Okazaki fragments together.

Semi-conserved replication

The principle of DNA replication where each new DNA molecule consists of one original parental strand and one newly synthesized strand.

Gel electrophoresis

A method for separation and analysis of macromolecules such as DNA, RNA, and proteins based on molecular size, charge, and shape.

Anode

The positive ($+$) pole in electrophoresis toward which molecules with a negative charge migrate.

Agarose

A linear carbohydrate (polysaccharide) extracted from seaweed used primarily for DNA and RNA separation.

Polyacrylamide gels (PAGE)

Gels formed from the polymerization of acrylamide and $N,N\text{'-methylenebisacrylamide}$ commonly used for protein separation.

SDS (sodium dodecyl sulphate)

An anionic detergent used in protein electrophoresis to denature proteins and impart a high-density negative charge.

Ethidium bromide

A DNA intercalator and potent mutagen that absorbs UV light ($300\text{ and }360\text{\thinspace nm}$) and emits in the visible range ($\text{yellow/orange } \thicksim 590\text{\thinspace nm}$).

Restriction enzymes (Endonucleases)

Enzymes that protect bacteria by cutting double-stranded DNA at specific nucleotide sequences known as recognition sites.

RFLP (Restriction fragment length polymorphism)

A method used to identify organisms by analyzing patterns derived from the cleavage of their DNA by restriction enzymes.

PFGE (Pulsed-Field Gel Electrophoresis)

A technique developed in 1984 as the gold standard for bacterial subtyping, suitable for large chromosomal DNA molecules ($> 15-20\text{\thinspace kb}$).

PCR (Polymerase Chain Reaction)

A technique conceived by Kary Mullis in 1983 that amplifies a small amount of template DNA into large quantities using repeated cycles of heating and cooling.

Taq polymerase

A thermostable DNA polymerase isolated from the bacterium $Thermus\text{ }aquaticus$ that can withstand high temperatures.

Primers

Short sequences of DNA (typically around $20\text{\thinspace bp}$) that provide a starting point for DNA synthesis by Taq polymerase.

dNTPs (deoxynucleotide triphosphates)

The building blocks (C, G, A, T) used in PCR for the synthesis of a new DNA strand.

Denaturation

The first step of PCR ($95\text{\thinspace }^{\text{o}}C$) where double-stranded DNA is heated to separate into single strands.

Annealing

The second step of PCR ($50-60\text{\thinspace }^{\text{o}}C$) during which primers base-pair with the template DNA.

Extension

The third step of PCR ($72\text{\thinspace }^{\text{o}}C$) where Taq polymerase copies the DNA strand by inserting complementary dNTPs.

Amplicons

The specific DNA products of the correct size produced at the end of a PCR cycle.

qPCR (Real-time PCR)

A quantitative PCR variation where product detection occurs through fluorescent signals during the reaction rather than at the end.

SYBR Green

An intercalating dye used in qPCR that emits a fluorescent signal when it binds to newly synthesized DNA.

TaqMan probes

Probes used in qPCR that rely on Förster Resonance Energy Transfer (FRET) and are cleaved by DNA polymerase to release a reporter signal.

RT-PCR (Reverse Transcriptase-PCR)

A technique used for gene expression analysis where mRNA is reverse transcribed into complementary DNA (cDNA).

ddPCR (Droplet Digital PCR)

An absolute quantification method for performing digital PCR based on water-oil emulsion droplet technology.

Sanger sequencing

A first-generation DNA sequencing method that produces fragments of different lengths marking the ends with fluorescent nucleotides.

Next generation sequencing (NGS)

A high-throughput sequencing approach that involves randomly fragmenting DNA into short sections (around $100\text{\thinspace bp}$) and sequencing vast numbers of reads simultaneously.

Ion Torrent sequencing

A semiconductor sequencing platform that detects nucleotide incorporation by measuring the flow of hydrogen ions released during synthesis.

Bridge amplification

A process used in Illumina sequencing where DNA molecules are amplified on a glass flow cell containing nanowells.