Bio Chem Ch 13

autoradiography

used to visualize imaged that have been exposed to oligonucleotides that have been radiolabeled

Florescence

EtBr slips between DNA bases and can be used as a stain

Nucleases

catalyze hydrolysis of phosphodiester backbone of nucleic acids

Endonuclease

cleavage in the middle

Exonuclease

cleavage at the ends of the molecule

Restriction Endonucleases

crucial role in recombinant DNA, cleaves double stranded DNA

• recognizes palindrome (read same from both ends)

• sticky ends joined by hydrogen bonding between complementary bases

• ligase reseals ends

EcoR1

Cleaves between A and G

Leaves sticky ends

Hae III

Cleaves between C and G

Leaves blunt ends

Gel Electrophoresis

separates based on size

Positive and negative end

No SDS

Recombinant DNA

molecules that contain DNA from two sources

Vectors

sources for getting recombinant DNA into cells

Phage

bacteria that infect viruses

Viral

Plasmid

small circular double stranded DNA molecule separate from chromosomal DNA

replicates independently

used for cloning

Bacterial

Plasmid- Selection

plasmid has selectable marker that indicated bacteria contains plasmid

• antibiotic resistance

Plasmid criteria

• origin of replication

• Selectable marker

• Multiple cloning site and restriction sites

Cloning with pUC plasmids

• cut foreign DNA with two different endonucleases

• Cut plasmid with endonucleases

Blue/white screening

• pUC plasmid contains lacZ gene

  • codes to cleave disaccharides

• contains X-gal that is colorless unless it interacts with B-galactosidase

• when foreign DNA is inserted, it disrupts the lacZ gene

No growth on petri dish

• no resistance

• no plasmid

Blue on dish

• resistance

• no DNA

• took up plasmid

No color

• took up plasmid

• DNA insterted

Expression Vectors

• Used to make proteins

• Needs origin of replication, multiple cloning sites, one selectable marker

• must be able to be transcribed with

  • promoter

  • ribosomal binding site

  • transcription termination sequence

Polymerase chain reaction (PCR)

• increases the amount of given DNA without cloning DNA

• heat DNA to separate

• 
  

PCR key component

taq polymerase from thermos aquatics

PCR primer issues

• needs the same GC content

• needs to be the same length

• cannot be complementary

DNA fingerprinting

shows only selected DNA sequences

• DNA digested with restriction enzymes and ran on agarose gel

• Uses southern blot: matches complementary DNA

  • Digest DNA with restriction endonucleases

  • Put in agarose gel electrohporases

  • Transferred to nitrocellulose paper

  • Incubate with DNA probe

  • Compare DNA to sample

Sequencing DNA

• uses dideoxy nucleotides

• missing OH at 3’, doesn’t allow DNA replication

• incorporates ddNTP on chain causes termination

• DNA sequence is mixed with oligonucleotide that serves as a primer for synthesis of complementary strand

• gel elctrophoresis is used to read strand