04-Exploring Genes

04-Exploring Genes - Exploring Genes Recombinant DNA...

Info iconThis preview shows pages 1–4. Sign up to view the full content.

View Full Document Right Arrow Icon
Exploring Genes Recombinant DNA technology: Manipulation of DNA Restriction enzymes to cut DNA. DNA ligases to join DNA fragments. Base pairing language, allowing complementary sequences to recognize and bind one another. Hybridization with complementary DNA or RNA probes for detection. Construction of new DNA combinations. Detection and amplification of particular sequences. Understanding of viruses to deliver DNA or RNA into hosts. Use of plasmids (accessory chromosomes in bacteria). Basic Tools Restriction enzymes: precise molecular scalpels. Blotting techniques: Southern and Northern blots to separate and characterize DNA and RNA, respectively. DNA sequencing. Solid-phase synthesis of nucleic acids for primers, fluorescent or radio- labeled DNA. Polymerase Chain Reaction (PCR): amplification of segments of DNA.
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Restriction Enzymes Restriction enzymes (aka restriction endonucleases): recognize specific base sequences in double-helical DNA and cleave, at specific places, both strands of a duplex. Found in many prokaryotes for cleaving foreign DNA. The cell’s own DNA recognition sites are methylated, thus not cleaved. Most restriction enzymes recognize specific sequences of 4~8 base pairs, and hydrolyze a phosphodiester bond in each strand in this region. These restriction sites almost always have pseudo twofold symmetry. The sequence is palindromic, or an inverted repeat, and the cleavage sites are symmetrically positioned. More than 100 restriction enzymes have been purified and characterized. Nomenclature: three letter abbreviation for the host, strain designation (if needed), roman numeral (if more than one enzyme from the same strain has been identified). Eco for Escherichia coli; Hin for Haemophilus influenzae; Hae for Haemophilus aegyptius. Fingerprinting DNA: a piece of DNA produced by cleaving with one restriction enzyme can be specifically cleaved into smaller fragments by another to yield particular patterns of fragments.
Background image of page 2
Gel Electrophoresis of DNA Electrophoretic mobility of DNA is inversely proportional to the logarithm of the number of base pairs. Polyacrylamide gels are used for 1000 base pairs. Agarose gels are used for larger fragments 20 kb. Extremely large nucleic acids can be separated on agarose gels by pulsed-field gel electrophoresis (PFGE). Differential stretching/relaxing of DNA as electric field is on/off. Visualization Radioactive DNA gels can be visualized by autoradiography. A gel can be stained with ethidium bromide, which fluoresces an intense orange when bound to double helical DNA. ( 50 ng) Southern Blotting (devised by Edwin Southern) A mixture of DNA is separated by electrophoresis, denatured to form ssDNA, transferred to a nitrocellulose sheet. The sheet is exposed to
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Image of page 4
This is the end of the preview. Sign up to access the rest of the document.

Page1 / 25

04-Exploring Genes - Exploring Genes Recombinant DNA...

This preview shows document pages 1 - 4. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online