BIOL 2500 - DNA Technology (Topic 10.1)

DNA technologies

techniques that obtain, amplify and manipulate DNA fragments

• genetic engineering and genomics are applications of DNA tech

blotting

an in vitro method to detect and quantify nucleic acid and proteins

southern blotting

works with DNA

• named after Edwin Southern

northern blotting

works with RNA

western blotting

works with protein

• sue antibodies to bind to specific proteins, which are detected by fluorescence or chemiluminescence

blotting steps

• gel electrophoresis

• molecule transfer to a membrane (special paper)

• visualized using a probe (usually radioactive) and autoradiography

restriction enzymes

form of recombinant DNA technology

• amplifies, maintains, and manipulates specific DNA sequence in vivo (inside the cell)

restriction mapping

uses specific enzymes to cut DNA at specific points resulting in double-stranded breaks

• much cheaper than DNA sequencing

• can obtain start and end sequences (need primers to do that in DNA sequencing)

• can compare DNA fragments across multiple organisms

• can be used to insert “cloned” DNA or PCR products

main steps of restriction mapping

• digest (cut) DNA w/ restriction enzymes (get fragments of varying lengths)

• separate fragments with electrophoresis

• visualize DNA (use stain or probe that fluoresces under UV light

polymerase chain reaction (PCR)

first step in sequencing the DNA of a DNA fragment (usually a gene of interest)

• developed in 1983 by Kary Mullis

3 main steps of PCR

1. DNA denaturation (1-2mins) - heat reaction mixture to 95* to allow double helix to unwind by breaking hydrogen bonds

2. primer annealing (1-2mins)

3. polymerase extension (3-5mins)

*repeat steps 1-3 over 25-35 cycles

thermocycler

machine that allows us to program the cycles, time, and temps. of the PCR conditions needed

quantitative PCR (qPCR)

• determines “real-time” amount of DNA produced during each PCR cycle

• uses SYBER Green instead of Ethidum bromide

• used to compare relative amounts of DNA molecules

reverse-transcription PCR (RT-PCR)

• used to detect, amplify and quantify mRNA

• mRNA is converted to cDNA (complementary DNA), which is double stranded, by a reverse transcriptase enzyme

• number of cDNA molecules is proportional to number of mRNA molecules so we can use this as an indication of expression levels