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