Applications of DNA Replication: PCR, DNA Sequencing, and STR Profiling

Vocabulary flashcards covering the principles, components, and procedures of PCR, gel electrophoresis, Sanger sequencing, Next-Generation Sequencing, STR profiling, and molecular mutation types.

Polymerase Chain Reaction (PCR)

A molecular biology technique first developed in $1983$ used for the exponential synthesis and amplification of a specific target DNA sequence, capable of amplifying DNA from a single cell in a few hours across $25-30$ cycles.

Oligonucleotide Primers (PCR)

Two short, synthetic single-stranded DNA sequences ($15-30$ nucleotides) that are complementary to the sequences flanking a target DNA region and provide the starting point for DNA synthesis on opposite strands.

Denaturation (PCR Step 1)

The initial step of a PCR cycle in which the reaction is heated to $90^\top\circ\text{C}-100^\top\circ\text{C}$ to separate double-stranded DNA into single strands.

Primer Annealing (PCR Step 2)

The step in a PCR cycle where the temperature is rapidly lowered to $30^\top\circ\text{C}-65^\top\circ\text{C}$ to enable short single-stranded primers to hybridize to their complementary sequences on target DNA.

Primer Extension (PCR Step 3)

The step in a PCR cycle where the temperature is raised to $72^\top\circ\text{C}$, enabling a thermostable DNA polymerase to synthesize new complementary DNA strands in the $5' \rightarrow 3'$ direction; duration depends on template length.

Taq Polymerase

A thermostable DNA polymerase used in PCR with an optimal temperature of $>70^\top\circ\text{C}$, high stability at $95^\top\circ\text{C}$, and high processivity in the $5' \rightarrow 3'$ direction, but lacking $3' \rightarrow 5'$ exonuclease proofreading activity.

Thermus aquaticus

A species of eubacteria that thrives in hot springs (such as those in Yellowstone National Park) from which Taq polymerase was originally isolated.

Gel Electrophoresis

A laboratory method in which an electrical current is applied to an agarose gel to separate negatively charged DNA fragments toward the positive pole based on size, where smaller fragments migrate faster and farther than larger ones.

Deoxynucleotide Triphosphate (dNTP)

A standard nucleotide precursor possessing a $3'\text{-OH}$ group on the deoxyribose ring that reacts to form phosphodiester bonds during DNA chain elongation.

Dideoxynucleotide Triphosphate (ddNTP)

A modified nucleotide that lacks a $3'\text{-OH}$ group on the sugar moiety, preventing the formation of a phosphodiester bond and terminating further DNA synthesis when randomly incorporated.

Dideoxy (Sanger) Sequencing

A sequencing technique combining single-stranded template DNA, a primer, DNA polymerase, a high concentration of all four dNTPs, and a low concentration of one specific ddNTP to produce fragments terminated at specific bases, which are then resolved on a gel.

Next-Generation Sequencing (NGS)

A high-throughput sequencing method (also called massive parallel or shotgun sequencing) that fragments DNA, attaches adapters, amplifies fragments simultaneously, and reads fluorescence additions to sequence entire genomes rapidly and affordably.

Contig

A continuous stretch of genomic sequence assembled by computer alignment of multiple overlapping DNA sequence reads generated during shotgun or NextGen sequencing.

Short Tandem Repeats (STRs)

Also called microsatellites; short repeated DNA sequence tracts distributed throughout the genome that naturally vary in repeat copy number among alleles and individuals.

TPOX

A human STR locus located on chromosome $2$ with the repeating sequence unit $\text{AATG}$.

CSF1PO

A human STR locus located on chromosome $5$ with the repeating sequence unit $\text{AGAT}$.

STR Profiling

A DNA fingerprinting method that combines PCR amplification of specific STR loci with gel electrophoresis to identify individuals by their unique fragment size patterns.

Base Substitution

A mutation in which one base pair is replaced by another without altering the total sequence length of the DNA.

Transition

A specific type of base substitution where a purine is replaced by another purine ($\text{A} \leftrightarrow \text{G}$) or a pyrimidine is replaced by another pyrimidine ($\text{C} \leftrightarrow \text{T}$).

Transversion

A base substitution in which a purine is swapped with a pyrimidine or vice versa ($\text{A} \leftrightarrow \text{C}$, $\text{A} \leftrightarrow \text{T}$, $\text{G} \leftrightarrow \text{C}$, or $\text{G} \leftrightarrow \text{T}$).

Indels

Mutations classified as insertions (the addition of $1+$ nucleotides) or deletions (the loss of $1+$ nucleotides) within a DNA sequence.