Genetic manipulation Differences between PCR and DNA replication

Genetic manipulation

Differences between PCR and DNA replication

PCR differences • Only uses small fragment of DNA – not whole genome • Uses Taq polymerase • Several changes in temperature – humans 37% • Several rounds of replications not just one • Doesn’t use a machine • No DNA helicase/gyrase • Not in cell

How does PCR reaction work – lab tech lost glasses and mixed up instructions? • Heat machine to 37 degrees • Add Taq RNA polymerase, primers, nucleotides, sample DNA • Add into machine – set for 10 cycles • First cycle 20, then 95, then 72 • Cut DNA with DNA ligase and run on gel with no dye. • Run gel for ten minutes

PCR cycle This Photo by Unknown Author is licensed under CC BY-SA

Gene sequencing – true or false statements • The Sanger method uses PCR method • The pyrosequencing method uses a gel • Pyrosequencing uses Ppi which reacts with other substrates to produce a photo of light • The Sanger method was very quick • Pyrosequencing happens in real time.

Sanger method This Photo by Unknown Author is licensed under CC BY-SA

Pyrosequencing • https: //en. wikipedia. org/wiki/File: How_Pyrosequencing_Works. svg

Genetic sequencing • Human genome • Comparing species • Evolutionary relationships • Variation between individuals • Predicting amino acid sequences • Synthetic biology – make insulin using bacteria, novel proteins, biofilms, biosensors, information storage.

Genetic engineering • Restrictions enzymes – bacterial enzymes that cut at certain bp • Vectors – carry genetic information into organism. Examples – plasmids, phages, Ti plasmid, virsus • Ligase – seals DNA into vector • Electroporation – use electricity to make membrane more porous.

How do I get gene – put into vector • m. RNA isolated – use reverse transcriptase – c. DNA, primers and polymerase PCR • Synthesis gene once know its nucleotide sequence • Amplify gene if code known using primers and PCR • Use probe to locate gene of genome – cut with restriction enzyme • Put into vector – plasmid, seal with ligase when cut with restriction enzyme.

Difficult bit – getting gene in • Heat shock treatment – cold zero degrees then 42 degrees with calcium chloride ions. • Electroporation – high voltage pulse • Electrofusion – electrical fields • Transfection – bacteriophage • Ti plasmid • Tungsten gun – add gold to gene and shoot it in.

How I do know if my bacteria got my gene • Plasmids can have A and B two antibiotic resistance genes, B gene with restriction site in where restriction enzyme cuts and FOREIGN DNA added so B gene will not work. • Put bacteria with plasmid into broth (growing stuff). • All bacteria without plasmid for A gene resistance die when put on petri dish with A antibiotic • Take bacterial colonies that did survive and put on plate with B antibiotic. • Bacteria which die on plate B have your gene. This Photo by Unknown Author is licensed under CC BY-SA

Examples and ethics • Ecoli – insulin, growth hormone – issues with antibiotic resistance ? • Plants Bt – Bt toxin pesticide – plant produce it kills insects – escape – super weeds ? • Soya beans – make roundup- superweeds ? • Golden rice – beta carotene – free licences to replace seeds • Silk – goats milk spiders web – welfare of animals • Virus – vaccine – oxford covid ? – reaction?