Biotechnology DNA Cloning

Compare/Contrast: PCR , Vector Cloning, & Sanger's DNA sequencing

PCR (Polymerase Chain Reaction) Steps

Vector Cloning 

Sanger's DNA Sequencing

Translation

1) Initiation

• mRNA transferred to the ribosomes
• Initiation factors (protein) assemble 
• Small ribosomal sub-unit - attaches to mRNA near start codon (AUG)
• Initiator tRNA - first to bind with codon (carrying methionine amino acid)
• mRNA
• Large ribosomal sub-unit - to form active ribosome
• 3 binding sites for tRNA 
• P (peptide) - contains tRNA with polypeptide attached
• A (amino acid) - contains tRNA with next amino acid added to polypeptide chain
• E (exit) - uncharged tRNA with no amino acid attached exits
• Moves from 5' to 3'

2) Elongation

• tRNA anticodons bind to mRNA codons in the 'A' site of the ribosome
• Initiating transfer RNA binds to the 'P' site
• tRNA recognizes the next codon and second amino acid moves to 'A' site
• Amino acid from tRNA joins amino acid by the tRNA that just entered 'A' site
• First transfer RNA is released
• Next transfer RNA moves to 'A' site and ribosome moves down one codon
• Second amino acid on tRNA in 'P' site transfers to third amino acid
• And Continues: Ribosome to move along mRNA and new amino acids are added to the growing polypeptide chain

3) Termination

• Terminates when mRNA reaches stop codon
• Stop Codons: UAA, UAG, UGA 
• Release factor (protein) cuts the polypeptide from last tRNA
  
  
  

Transcription

1) Initiation

• Transcription Factors assemble on the promoter region (TATA box)
• RNA polymerase II binds to the transcription factors (transcription initiation complex)

2) Elongation

• Moving from 5' to 3', RNA polymerase synthesizes a strand of pre-mRNA that is complimentary to the template strand of DNA
• Template strand (antisense strand)- the sequence of DNA that is copied 
• Coding strand (sense strand)- the sequence that corresponds with 
• Pre-mRNA strand, Thymine is replaced with Uracil 

3) Termination

• RNA polymerase II reaches the terminator sequence (AAUAA), transcription stops
• Pre-mRNA is removed
• Pre-mRNA protected by G cap (guanine) on the 5' side and poly A tail on the 3' side
• RNA splicing 
• Introns (non-coding regions) - are removed from pre-mRNA
• Exons (coding regions) - join to form the mature mRNA
• snRNA + snRNP(proteins) - recognizes regions where exons and introns meet
• snRNP interact with other proteins = larger spliceosome complex that removes introns
• mRNA strand is complete and ready to transport to the ribosomes (factory)

DNA Replication

The Process of DNA Replication:
1) Initiation

• Helicase - unwinds the double stranded DNA helix 
• Single-Strand Binding Proteins - react with single stranded DNA to stabilize it and keep the DNA strands separated
• Gyrase - relieves tension in the DNA from unwinding process
• Primase (RNA Polymerase)
           - creates RNA primers (~10 primers; according to parent strand)
           - initiates Polymerase III to form complementary strand 

2) Elongation

• DNA Polymerase III - attaches to a primer and adds nucleotides to 3' end of the new DNA strand
• 2 types of strands in each strand:
  - Leading Strand - from 5' --> 3' into replication fork
  - Lagging Strand - from 5' --> 3' away from replication fork; replicates in short segments (Okazaki Fragments)
• DNA Polymerase I - replaces Polymerase III when it reaches the primer and replaces the RNA primer with correct DNA sequence

3) Termination

• DNA ligase
           - attaches and forms phosphodiester bonds  
           - connects Okazaki Fragments

Note: 
- semi-conservative - one parent strand & one daughter strand in the replicated DNA
- new DNA strand can only elongate in the 5' --> 3' direction