Chapter 13

Restriction Endonucleases

Enzymes that recognize short DNA sequences and cleave DNA phosphodiester bonds, producing specific double-strand fragments with terminal 5’-phosphate.

Sticky ends

Result of staggered cuts made by restriction endonucleases, creating overhanging ends in DNA fragments.

Blunt ends

Result of straight cuts made by restriction endonucleases, producing DNA fragments with no overhangs.

Sanger Method

A DNA sequencing technique involving controlled interruption of enzymatic synthesis, generating DNA fragments of varying lengths.

Polymerase Chain Reaction (PCR)

A method to amplify DNA in vitro, involving denaturation, annealing, and extension steps.

Type I Restriction Endonucleases

Large, multi-subunit proteins that cleave DNA at random sites and are ATP- and SAM-dependent.

Type II Restriction Endonucleases

Enzymes that cleave DNA at specific distances from recognition sites, not requiring ATP or SAM.

Type III Restriction Endonucleases

Large proteins that cleave DNA at random sites near recognition sequences, ATP-dependent, and SAM-activates.

Type IV Restriction Endonucleases

Enzymes targeting only methylated DNA for cleavage.

DNA Sequencing Process

Involves four separate sequencing reactions with varying lengths of DNA fragments terminated with dideoxynucleotides.