PCR, Sanger Sequencing and Vector Cloning (Compare and Contrast)

PCR (Polymerase Chain Reaction)

·      Definition: fast and inexpensive technique used to amplify small and targeted segments of DNA to produce million or billions of copies

·      Requirements: DNA template, taq polymerase, dNTP (dATP, dCTP, dGTP, dTTP), primers (2 known sequences)

·      Steps: Denaturation> Annealing> Extension

·      Denaturation: At 95%, the heat denatures the DNA into two single strands by breaking hydrogen bonds

·      Annealment: Cooled to 55%, primers bind to the 3' end of the target DNA at both strands

·      Extension, At 72%, nucleotides are added by taq polymerase

·      Applications: DNA fingerprinting,

Sanger Sequencing

·      Definition: most popular method of DNA sequencing developed by Fred Sanger

·      Requirements: DNA template, dNTP, DNA polymerase, primer (1 sequence), and ddNTP (fragments of various lengths will be synthesized)

·      Dideoxynucleotides are essentially the same as nucleotides except they contain a hydrogen group on the 3’ carbon instead of a hydroxyl group (OH).

·      Steps: Denaturation> Annealing> Extension (ddNTPs) > Gel electrophoresis

·      Gel electrophoresis is used to determine the DNA sequence (Shorter fragments to Longer fragments synthesized by ddNTP)

Vector Cloning

·      Definition: Makes use of a cloning vector (plasmid), a DNA molecule that carries foreign DNA into a host cell, replicates inside a bacterial (or yeast) cell and produces many copies of itself and the foreign DNA

·      Requirements: restriction enzymes(cuts DNA molecules at specific locations which must produce sticky ends), plasmid, gene of interest, bacteria, ligase 

·      Steps: Recombinant DNA (Foreign DNA to plasmid using restriction enzymes and ligase)>Transformation(DNA to bacteria)> Cloning > Purification

·      Bacteria are used as host cells because they grow rapidly and DNA can be easily isolated and reintroduced into their cells 

·      Application: prepare many copies of the gene

PCR & Sanger Sequencing

• ·      PCR is used in Sanger Sequencing.
• ·      PCR uses dNTP(makin copies of DNA) while Sanger Sequencing uses ddNTP(sequencing purposes)

PCR & Vector Cloning

• ·      Both results to making copies of DNA
• ·      Vector Cloning takes longer than PCR
• ·      Vector Cloning uses plasmid, bacteria cells to make copies of gene while PCR uses primers, taq polymerase and dNTPs.

Sanger Sequencing and Vector Cloning

• ·      Sanger Sequencing is used to sequence DNA while Vector Cloning is used to make copies of gene of interest using transformation and bacteria.

PCR, Sanger Sequencing and Vector Cloning

• ·      All are processes and applications in Biotechnology.
• ·      All of them requires gene of interest for different applications and purposes.