PCR and DNA Replication

PCR (Polymerase Chain Reaction)

A technique used to amplify a specific region of DNA, producing billions of copies.

Purpose of PCR

To make many copies of a specific DNA region for analysis.

PCR principles

PCR repeatedly separates DNA strands, allows primers to bind, and uses DNA polymerase to synthesize new strands, doubling the target DNA approximately each cycle.

PCR template DNA

The original DNA containing the region to be amplified; in this experiment, genomic DNA.

PCR primers

Short, single-stranded DNA sequences that bind to opposite ends of the target region and provide a starting point for DNA synthesis.

Primer design

Determines which DNA region is amplified and the length of the PCR product. The amplified region lies between the primers.

PCR product

The amplified DNA fragment whose sequence and length are determined by the primers.

DNA polymerase

An enzyme that synthesizes new DNA strands by adding nucleotides to the growing strand's 3′ end.

Thermostable DNA polymerase

A heat-resistant DNA polymerase used in PCR that survives repeated high-temperature cycles and works best around 72°C.

Taq polymerase

A commonly used thermostable DNA polymerase originally isolated from a heat-loving bacterium.

dNTPs

The four DNA building blocks used to synthesize new DNA: dATP, dCTP, dGTP, and dTTP.

Mg²⁺

A required cofactor that helps DNA polymerase function.

PCR Master Mix

A mixture containing thermostable DNA polymerase, dNTPs, Mg²⁺, salts/buffer, and water. Primers and template DNA are added separately in this experiment.

PCR denaturation

The high-temperature step, usually 95°C, that separates double-stranded DNA by breaking hydrogen bonds between complementary bases.

PCR annealing

The cooling step, usually around 55°C in this experiment, that allows primers to bind to complementary DNA sequences.

PCR elongation (extension)

The step, usually around 72°C, when DNA polymerase extends the primers and synthesizes new DNA strands in the 5′ → 3′ direction.

PCR enzyme activation

The initial heating step that activates the DNA polymerase used in the reaction; in this experiment, 95°C for 2 minutes.

PCR final extension

An additional extension step at 72°C for 10 minutes that allows incomplete DNA strands to finish extending.

PCR storage

Holding the reaction at 4°C to preserve the products until they are retrieved.

PCR cycle

One round of denaturation, annealing, and elongation. Repeated cycles amplify the target DNA.

PCR program

The sequence of temperatures and times used to carry out PCR.

PCR denaturation temperature and time

95°C for 30 seconds per cycle in this experiment.

PCR annealing temperature and time

55°C for 30 seconds in this experiment.

PCR elongation temperature and time

72°C for 1 minute 40 seconds in this experiment.

Number of PCR cycles

This experiment repeats the denaturation, annealing, and elongation steps 34 times after the initial activation step.

Exponential amplification

The rapid increase in target DNA copies during PCR. Ideally, the amount of target DNA doubles each cycle.

PCR product after nn cycles

Ideally, 2n2^n double-stranded copies are produced from one starting double-stranded target molecule after nn cycles, assuming perfect efficiency.

PCR first-cycle products

Early products can extend beyond the intended target region because polymerase copies the available template to its end.

PCR specific-length products

Products with both ends defined by the primers accumulate during later cycles and become the predominant product.

DNA replication in cells

The process by which a cell copies its entire genome before cell division.

Similarity between cellular DNA replication and PCR

Both use a DNA template, primers, DNA polymerase, and nucleotides to synthesize complementary DNA in the 5′ → 3′ direction.

Main difference between PCR and cellular DNA replication

Cellular replication copies the entire genome using cellular machinery, while PCR amplifies a specific DNA region using repeated temperature changes and thermostable DNA polymerase.

Initiation in cellular DNA replication

Replication begins at origins of replication. Helicase separates DNA strands, and gyrase helps relieve supercoiling in bacteria.

Initiation in PCR

Heating to approximately 95°C separates DNA strands without requiring helicase.

Helicase

The cellular enzyme that unwinds double-stranded DNA by separating its strands.

Gyrase

A bacterial enzyme that relieves supercoiling and torsional strain ahead of the replication fork.

Primase

The cellular enzyme that synthesizes short RNA primers to initiate DNA synthesis.

Priming in cellular DNA replication

Primase makes RNA primers that provide a starting point for DNA polymerase.

Priming in PCR

Synthetic DNA primers are added to the reaction and bind to complementary sequences during annealing.

RNA primer

A short RNA sequence synthesized by primase during cellular DNA replication.

DNA primer

A short DNA sequence used in PCR to initiate DNA synthesis; it becomes part of the amplified product.

Leading strand

The new DNA strand synthesized continuously toward the replication fork in the 5′ → 3′ direction.

Lagging strand

The new DNA strand synthesized discontinuously away from the replication fork in short Okazaki fragments, each made in the 5′ → 3′ direction.

DNA polymerase III

The main DNA-synthesizing enzyme in bacterial cellular replication.

DNA polymerase I

A bacterial enzyme that removes RNA primers and replaces them with DNA during replication.

Okazaki fragments

Short DNA segments synthesized on the lagging strand during cellular DNA replication.

DNA ligase

An enzyme that joins adjacent DNA fragments by sealing breaks in the sugar-phosphate backbone.

Leading and lagging strands in PCR

PCR does not have distinct leading and lagging strands because it does not use a replication fork; each template strand can be copied by extension from its primer.

Primer removal in cellular replication

DNA polymerase I removes RNA primers and replaces them with DNA; DNA ligase seals the remaining breaks.

Primer removal in PCR

PCR primers are not removed during amplification; they become part of the newly synthesized DNA strands.

Termination in cellular DNA replication

Replication ends when the DNA has been copied, such as when replication forks meet or reach termination regions.

Termination in PCR

DNA synthesis stops when polymerase reaches the end of its template or when the temperature changes.

Semiconservative replication

Each newly replicated DNA molecule contains one original strand and one newly synthesized strand.

Cellular DNA replication product

A complete copy of the genome is produced, with each daughter DNA molecule containing one parental strand and one new strand.

PCR product length

Determined by the positions of the primers, specifically the region between their 5′ ends on the amplified strands.

Restriction enzyme

An enzyme that recognizes a specific DNA sequence and cuts DNA at or near that sequence.

Restriction digest

A procedure in which restriction enzymes cut DNA at specific recognition sequences.

Restriction fragment length polymorphism (RFLP)

A difference in DNA fragment lengths resulting from genetic variation that changes restriction enzyme cutting patterns.

RFLP analysis

A method that uses restriction enzyme digestion and DNA fragment sizes to identify genetic differences.

Single-nucleotide polymorphism (SNP)

A genetic variation involving a change in one nucleotide at a particular position in the DNA sequence.

Wild-type (wt) allele

The reference or commonly occurring allele; in this experiment, it contains the restriction enzyme recognition site.

Loss-of-function allele

A genetic variant that reduces or eliminates the function of a gene product. In this experiment, the SNP disrupts the restriction enzyme recognition site.

TAS2R38 gene

A gene on chromosome 7 that encodes a bitter-taste receptor and is used in this experiment to study genetic variation.

Purpose of the TAS2R38 experiment

PCR amplifies a region containing a SNP, then restriction enzyme digestion distinguishes alleles based on whether the enzyme can cut the DNA.

TAS2R38 PCR product

An approximately 220-base-pair DNA fragment containing the SNP of interest.

Restriction enzyme digestion of the wild-type TAS2R38 fragment

The enzyme cuts the approximately 220-bp fragment into fragments of approximately 177 bp and 44 bp.

Restriction enzyme digestion of the loss-of-function TAS2R38 fragment

If the SNP disrupts the recognition site, the enzyme cannot cut the fragment, so it remains approximately 220 bp.

Uncut TAS2R38 fragment

Approximately 220 bp; indicates that the tested DNA fragment was not cut at the target restriction site.

Cut TAS2R38 fragments

Approximately 177 bp and 44 bp; indicate that the restriction enzyme recognition site was present and the DNA was cut.

Agarose gel electrophoresis

A technique that separates DNA fragments according to size by applying an electric field across an agarose gel.

Why DNA moves toward the positive electrode

DNA has a negatively charged phosphate backbone, so it migrates toward the positively charged electrode.

How agarose gel separates DNA

The agarose forms a porous matrix that slows larger fragments more than smaller fragments.

Which DNA fragments move fastest through agarose?

The smallest DNA fragments move fastest and generally travel the farthest in a given amount of time.

Expected migration order of TAS2R38 fragments

The 44-bp fragment migrates fastest, followed by the 177-bp fragment, then the 220-bp fragment.

DNA fragment size

The length of a DNA fragment, commonly measured in base pairs (bp) for double-stranded DNA.

Base pair (bp)

A unit of DNA length representing one paired set of complementary nucleotides in double-stranded DNA.

Homozygous wild-type genotype

Both copies carry the wild-type allele; digestion produces the cut fragments, approximately 177 bp and 44 bp.

Homozygous variant genotype

Both copies carry the SNP allele that disrupts the restriction site; digestion produces an uncut fragment of approximately 220 bp.

Heterozygous genotype

One copy carries the wild-type allele and the other carries the variant allele; digestion produces both the 220-bp uncut fragment and the 177-bp and 44-bp cut fragments.

Interpreting RFLP gel bands

Band sizes reveal whether the DNA was cut, helping identify the alleles present. The 44-bp band may be faint or difficult to see.

Why PCR is useful before RFLP analysis

PCR makes enough copies of the specific region containing the SNP for restriction digestion and gel analysis.