PCR

PCR

1. PCR is used to amplify (make many copies of) a specific part of a DNA or RNA sequence

2. Short "primers" are synthesized so that they are able to base-pair with specific regions on the template/target

3. DNA polymerase extends the primer based on the sequence of the template

PCR 1

PCR is used to amplify (make many copies of) a specific part of a DNA or RNA sequence

PCR 2

Short "primers" are synthesized so that they are able to base-pair with specific regions on the template/target

PCR 3

DNA polymerase extends the primer based on the sequence of the template

PCR rounds

15-30, most of the product is the sequence between the primers

Process

1. use heat to denature DNA

   1. strands held together by noncovalent interactions (hydrogen bonds I think), destabilize over high heat

2. primer is combined, anneal/base pair to a specific spot on sequence

   1. 15-25 nucleotides long

3. primers are extended by DNA polymerase

   1. will be reverse complement

4. 15-30 cycles, accumulate sequences of specific length

Process 1

use heat to denature DNA strands

• held together by noncovalent interactions (hydrogen bonds I think), destabilize over high heat

Process 2

primer is combined, anneal/base pair to a specific spot on sequence

• 15-25 nucleotides long

Process 3

primers are extended by DNA polymerase

• will be reverse complement

Process 4

15-30 cycles, accumulate sequences of specific length

Applications of PCR

• Amplify an allele to express it in another system

  • transgenic organisms

  • isolate the encoded RNA or protein for biochemical studies

• Detecting SNPs

  • hybridize PCR products to a microarray

  • amplify using primers that base-pair to one SNP variant but not another

• Detecting insertions/deletions

  • primers that flank a variable region —gel electrophoresis to detect the size of that region in each individual/sample

• Amplify a specific region for sequencing

Applications of PCR 1

• Amplify an allele to express it in another system

  • transgenic organisms

  • isolate the encoded RNA or protein for biochemical studies

Applications of PCR 2

• Detecting SNPs

  • hybridize PCR products to a microarray

  • amplify using primers that base-pair to one SNP variant but not another

Applications of PCR 3

• Detecting insertions/deletions

  • primers that flank a variable region —gel electrophoresis to detect the size of that region in each individual/sample

Applications of PCR 4

• Amplify a specific region for sequencing

PCR

A laboratory technique used to amplify (make many copies of) a specific DNA or RNA sequence.

Amplify (in PCR)

To generate many identical copies of a specific DNA or RNA sequence.

Primers in PCR

Short, synthetic oligonucleotides (15–25 nucleotides long) that are designed to base pair only with a specific region of the template DNA being amplified.

Primer design

Primers are complementary to the ends of the target region of DNA so that amplification occurs only between them.

DNA polymerase (PCR)

The enzyme that extends primers by adding nucleotides complementary to the template strand, creating a new DNA strand.

Template DNA

The original DNA strand that provides the sequence to be copied during PCR.

PCR Step 1: Denaturation

Heat is applied to separate the two strands of DNA by breaking non-covalent interactions between them.

PCR Step 2: Annealing

Cooling allows primers to base pair (anneal) to the specific complementary sequences on the single-stranded DNA templates.

PCR Step 3: Extension

DNA polymerase extends the primers, adding nucleotides complementary to the template strand, creating new DNA strands.

Cycle of PCR

One full round of denaturation, annealing, and extension. Each cycle doubles the number of DNA molecules.

Length of primers (typical)

Usually between 15–25 nucleotides long, though in the diagram shown they were simplified to 5 nucleotides.

After 1 cycle of PCR

Two new DNA products are made in addition to the original templates.

After 2 cycles of PCR

products are defined by primers on one side and template on the other

After 3 cycles of PCR

products appear that are exactly the distance from one primer to the other — the specific amplified region.

After 4 cycles of PCR

More products accumulate that are precisely the primer-to-primer length; these become the dominant form.

PCR product specificity

Over many cycles (15–30+), the majority of PCR products are exactly the length between the two primers. Longer products exist but are not predominant.

Typical PCR cycle count

Usually between 15 and 30 cycles (sometimes more), generating millions of copies of the target sequence.

Main product of PCR

DNA fragments that are exactly the length from primer to primer, representing the specific amplified region.

Application of PCR 1

Allele amplification
PCR can amplify an allele so it can be expressed in another system (e.g., cloning into a vector).

Application of PCR: Cloning

PCR products can be inserted into vectors downstream of promoters to allow protein expression in systems like bacteria or yeast.

Application of PCR: Protein expression

PCR products cloned into expression vectors can be used to produce proteins for biochemical study.

Application of PCR: Detecting SNPs

PCR can detect single nucleotide polymorphisms (SNPs) by: (1) hybridizing PCR products to microarrays, or (2) using SNP-specific primers that only bind one variant.

Application of PCR: Detecting insertions/deletions

Primers flanking variable regions can amplify fragments of different lengths, which can then be analyzed (e.g., gel electrophoresis).

Application of PCR: DNA sequencing

PCR can amplify a specific region of DNA (e.g., suspected mutation site) so it can be sequenced without sequencing the entire genome.

PCR amplified sequence

Defined entirely by the two primers; the final product is only the distance from one primer to the other.

NTPs in PCR

Nucleoside triphosphates (building blocks) that DNA polymerase uses to synthesize new DNA strands during extension.