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.

Translation

Translation

Synopsis: Translation is the process by which a protein is synthesized from the information contained in messenger RNA (mRNA).

Characters: tRNA, small and large ribosomal unit, codons, start and stop codons, anticodons, rRNA, ribosome

Plot: Initiation, Elongation, Termination

ACT I: Initiation

• mRNA, the tRNA with the first amino acid (methionine)and two ribosomal subunits(small and large) gather together.
• Small ribosomal subunits bind with the mRNA and the tRNA that has the methionine amino acid and then binds to the start codon (AUG).
• Initiation factors bring large subunits such as the tRNA to take over the P site.

Act 2 - Elongation 

• Anticodons of tRNA molecules bind to mRNA codons in the A site of the ribosome. 
• rRNA catalyzes peptide bond formation among the polypeptide located in the P site with the amino acid in the A site.
•  The tRNA molecule with the polypeptide is moved from the A site to the P site (translocation), and codons are matched with their respective anticodons in the 5' to 3' direction. After that, it exits at E site.
• Process is repeated by entering a new codon at site.

Act 3 - Termination 

• Translation continues until ribosome reaches 1 of the 3 stop codons (UGA, UAG and UAA).
• Termination proteins bind to the ribosome and a release factor cuts the polypeptide chain from the last tRNA. 
• Large and small subunit separates as ribosome released from mRNA.

VIDEO

DNA Transcription

DNA Transcription

Synopsis: DNA Transcription is the synthesis of RNA from a DNA template where the code in the DNA is converted into a complementary RNA code.
Setting: Promoter Region
Characters: Antisense or template strand, Sense strand or coding strand, RNA polymerase II, transcription factors, 5' cap, poly-A tail, introns, exons
Plot: Initiation, Elongation, Termination

ACT I: Initiation

• Transcription factors (TFs) identify and bind to the promoter region. (TATA Box)
• RNA polymerase II binds to the transcription factors in DNA and forms a transcription initiation complex.

ACT II: Elongation

• RNA Polymerase II synthesize a strand of pre-mRNA (complementary to template strand) by adding nucleotides to the 3' end of the growing pre-mRNA. Transcription goes from upstream towards downstream of the DNA strands in a 5' to 3' direction.
• Pre-mRNA is anti-parallel with the template strand (Antisense), and it's similar with the coding strand (Sense) except U(pre-mRNA) and T(DNA strand). Uracil replaces thymine within the pre-mRNA.

ACT III: Termination

• RNA Transcription stops when it reaches the terminator region (AAUAAA).
• The pre-MRNA will undergo modification to mRNA.
•  A 5' cap which has an altered form of guanine is added to the pre-mRNA strand for protectionand to indicate to ribosomes where to start. Also, a poly-A-tail is added to the 3' end (series of adenine nucleotides). 
• RNA splicing occurs  where introns (non-coding regions) are removed from the transcript by making a loop, leaving only exons (coding segments), which is done by the splicosome, consisting of snRNPs and snRNA.
• As a result, the mRNA strand is complete and ready for transport to the ribosomes.

• Video