PCR, Vector Cloning, and Sanger's Sequencing

-producing DNA

PCR: Polymerase Chain Reaction

-best method for preparing large quantities of a particular gene or other DNA sequence (billions of copies of a section of DNA made in hours)

-quicker and more selective than vector cloning when source of DNA is scanty or impure 

-requires: DNA template, dNTP (dATP, dCTP, dGTP, dTTP), taq polymerase (heat resistant), primers (2 known sequences)   

-3 steps: denaturation, annealing, extension

-Denaturation: uses heat (more than 90 degrees Celsius) to separate double stranded DNA

-Annealing: 2 primers (one for each single strand of DNA) and taq polymerase attach to DNA strand (tube is cooled in this step: 40-60 degrees Celsius)

-Extension: bases are added to primers to create new strand of complementary DNA (temperature is increased again in this step to about 72 degrees Celsius) 

-application: forensic analysis (example: isolating samples of DNA  from blood at a crime scene), determining ancient evolutionary relationships (DNA from mummies and fossil tissue) 

Vector Cloning: 

-insertion of foreign gene into bacterial plasmid 

-requires restriction enzymes that cut DNA molecules at specific locations (needs to recognize short DNA nucleotide sequences and cut at specific point in sequence, must produce sticky ends), ligase 

-long process  

-commonly uses bacteria as host cells because they grow rapidly and DNA can be easily isolated and reintroduced into their cells 

-DNA screened after amplified to get desired gene 

-potential uses: production of protein product or to prepare many copies of the gene 

Sanger's Sequencing

-similar to PCR 

-requires: DNA template, dNTP, polymerase (normal polymerase- no heat), primer (1 known sequence), and ddNTP (di deoxy (O, die!))

-ddNTP makes sure that fragments of various lengths will be synthesized 

-gel electrophoresis is used to interpret the nucleotide sequence (reading order of fragments) 

-single stranded DNA with unknown sequence acting as template + DNA polymerase +dNTPS + radioactively labeled primer + ddNTPS ---> gel electrophoresis ----> auto-radiography ----> sequence of new strand 

Initiation, Elongation, and Termination of Translation

Translation:

Initiation:
-In the ribosome, mRNA and tRNA come together
-In the ribosome, there is the big subunit and the small subunit
-Bigger subunit joins the smaller subunit, the first a.a. tRNA, and mRNA (initiation complex)
-The start codon for the protein is AUG (methionine)

Elongation:
-addition of codons/amino acids that will eventually make up the polypetide (final result)
-three sites: APE (A-entrance, P-prison, G-exit)
-amino acids can bind in the A-site and P-site
-initiating tRNA binds to the P site, then binds to an amino acid brought by the next tRNA (in the A-site)
-After the amino acids bind, the first initiating tRNA is released from the P site (prison)

Termination:
-The process of amino acid binding is continued until a stop codon is brought to the A site on the mRNA
-These stop codons are: UAA, UAG, and UGA

Initiation, Elongation, and Termination of Transcription

Initiation: 

Main 'characters': Transcription factors, promoter region (TATA box), polymerase 2 

-Transcription Factors (TFs) bind to the promoter region (eukaryotes need other proteins to recruit polymerase) 

-TATA box (found upstream) is the site to bind for TFs 

-Once binding is complete, poymerase 2 begins to read TF and transcribes on a complementary strand 

Elongation:

Main 'characters': Polymerase 2, coding strand, template strand 

-Elongation in transcription also occurs in a 5' to 3' direction 

-The strand that is very similar to the RNA transcript is the coding/sense strand (the opposite is the template/antisense strand) 

-RNA is anti parallel to the templat/antisense strand 

-During transcription, Thyamine (T) is replaced by Uracil (U) (Love U and not T(ea))

Termination: 

Main 'characters': AAAUAAA, intron, exon, G-cap and poly-a tail

-Transcription stops when polymerase 2 reaches the terminator AAAUAAA(downstream) 

-pre mRNA can't be sent into the cytoplasm (water=oxygen filled; oxygen=bully)

-G-cap and poly A-tail put on ends of the strands to protect from bullies

-intron=junk DNA to be cut out