Lecture 22 - Recombinant DNA

What is recombinant DNA?

the joining of DNA molecules, usually from different biological sources that are not found together in nature

Procedure for producing recombinant DNA

• generating specific DNA fragments using restriction enzymes

• joining these fragments with a plasmid

• transferring the recombinant DNA molecule to a host cell to produce many copies that can be recovered from the host cell

clones

recovered copies of a recombinant DNA molecule

• used to study the structure and orientation of the DNA

restriction enzyme

binds to DNA at a specific recognition sequence (restriction site) and cleaves the DNA to produce restriction fragments

Insulin

first protein to be chemically synthesized and produced by DNA recombinant technology

Recognition Sequences

• most sequences exhibit a form of symmetry described as a palindrome, and restriction enzymes cut the DNA in a characteristic cleavage pattern

  • mostly 4-6 nucleotides long

  • some contain eight or more nucleotides

• DNA ligase joins restriction fragments covalently to produce intact DNA molecules

DNA Vectors

Vectors (also called plasmids) are carrier DNA molecules that can replicate cloned DNA fragments in a host cell

• must be able to replicate independently and should have several restriction enzyme sites to allow insertion of a DNA fragment

• should carry a selectable gene marker to distinguish host cells that have taken them up from those that have not

DNA Vector Structure

• extrachromosomal double-stranded DNA molecule that replicates independently from the chromosomes within bacterial cells

• circular (in bacteria)

• range from 1kb to over 200kb

• replicate autonomously

• many carry antibiotic-resistance genes, which can be used as selectable markers

Transformation

Introduces plasmids; achieved through:

• using calcium ions and brief heat shock to pulse DNA into cells

• electroporation, which uses a brief but high-intensity pulse of electricity to move DNA into bacterial cells

DNA Expression Vectors

• engineered to express a gene of interest to produce large quantities of the encoded protein in a host cell

• available for both prokaryotic and eukaryotic host cells

• plant and animal cells can serve as hosts for recombinant DNA, in addition to bacteria and yeast

Polymerase Chain Reaction (PCR)

• copies a specific DNA sequence through in vitro reactions that can amplify target DNA sequences present in very small quantities

• rapid method of DNA cloning that eliminates the need to use host cells for cloning

Taq Polymerase

introduced by Randall Saiki in 1986

• DNA polymerase from Thermus aquaticus is used for PCR because it is heat-stable

• lacks proofreading activity, so errors are introduced into the amplified DNA at low but significant frequencies

• amplifies fragments of DNA larger than a few base pairs inefficiently

PCR Process

• double-stranded DNA to be cloned is put in a tube with:

  • DNA Polymerase, Mg²⁺, four deoxyribonucleoside triphosphates

• requires two oligonucleotide primers, one complementary to the 5’ end of one strand of the target DNA to be amplified and another complementary to the 3’ end of the other strand

• 3 steps of PCR:

  • denaturation, primer annealing, and extension

  • repeated over and over using a thermocycler to amplify the DNA exponentially

• DNA strand is doubled in each cycle, and the new strands along with the old strand serve as templates in the next cycle