Lecture 17 - DNA Technology

DNA technology

• techniques for sequencing and manipulating DNA

• many involve DNA hybridization - complementary base pairing

polymerase chain reaction (PCR)

• method of producing billions of copies of specific DNA sequence in short time (hours)

• important when studying genes - any specific gene is a tiny fraction of total DNA

• components:

  • template DNA

  • dNTPs

  • primers

  • Taq polymerase

• SEQ the process

template DNA

sample containing DNA sequence you want to amplify

dNTPs

deoxyribonucleotide triphosphate - As, Ts, Cs, and Gs

primers

• short (15-20 base) DNA fragments with specific sequences

• match each end of your target DNA sequence - set ends of amplified region

Taq polymerase

DNA polymerase from Thermus aquatics, bacteria from hot springs

process of PCR

3 steps repeated in 30-40 cycles

• denature

  • heat to ~90*C

  • breaks h-bonds

  • separates double helix (helices activity via heat)

• annealing

  • cool to ~40-65*C

  • primers bind with complementary sequences on template DNA

• extension

  • ~72*C

  • Taq polymerase replicates DNA from 3’ end of primers

  • resulting DNA molecules are the templates for the next round

thermal cycler

• used in process of PCR

• machine that can precisely modulate temperature of mixture

gel electrophoresis

• technique for separating nucleic acid molecules by size

• often used to confirm PCR results

• method:

  • make polysaccharide gel - porous

  • load DNA into wells

  • pass electrical current through gel (DNA moves - to +)

  • smaller DNA fragments move faster than larger because it doesn’t get hung up on the matrix

• uses:

  • confirmed successful PCR

  • DNA sequencing

  • DNA profiling/barcoding

  • disease diagnosis

applications of DNA technology

• DNA sequencing

• species diversity

• gene therapy

DNA sequencing

• chain termination sequencing most common

• PCR + gel

• basic idea:

  • copy the template DNA, but make fragments of different lengths

  • run them on a gel to separate by size

  • read the sequence from shortest to longest fragment

species diversity

• hard to count every species in a community, esp tiny(many under microscope) or rare ones

• solution: DNA barcoding

  • sequence one short, standard region of the genome and use it as an ID tag for the organism

• need only a tiny tissue sample

• e.g.

  • Animals: cytochrome c oxidase

  • Other groups use other genes (e.g., rubisco for plants, rRNA for others)

gene therapy

• in use:

  • e.g. retrovirus delivers a correct copy of the gene into retina cells as a treatment for a genetic form of blindness

• potential future:

  • CRISPR: tool to silence or insert specific genes → …