Basics in molecular biology •Transcription
–Intron
–Poly A tail
•Translation
–Eukaryotes: Nucleus/endoplasmic reticulum
–Prokaryotes: Coupled transcription-translation •Commonly used enzymes
–Restriction endonucleases
–DNA polymerase I
–DNA polymerase I large fragment (Klenow)
–Polynucleotide kinase
–Reverse transcriptase
–Terminal deoxynucleotidyl transferase
–Alkaline phosphatase
–RNA polymerase
–DNA ligase
–RNA ligase
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•Restriction enzymes
–Class of bacterial enzymes that cut DNA at specific sites
–Restriction: In bacteria their function is to destroy foreign DNA such as that of bacteriophages and to restrict viral host range.
–Restriction modification system.
–HindIII, 1970. Nobel Prize in 1978.
–>3000 enzymes studied in detail and >600 commercialized.
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–Differ from other nucleases: recognize and cleave a specific DNA sequence. –Over 250 sequence specificities have been discovered.
•Type II restriction enzymes
–Type I and Type III restriction endonucleases.
–Subunit composition, cleavage position, sequence-specificity and cofactor-requirements.
–Functions, conditions, recognition sequences and cutting sites of three types of restriction enzymes.
–Are the classic experimental tools.
–Have very specific recognition and cutting sites.
•Recognition sites
–Generally are 4, 6, or 8 bp in length.
–Most sites are palindromic sequences
–Sequences reads the same in a 5’--->3’ direction on each strand.ilvs
–Examples of cleavage sites
•4 bp recognition sites: HaeIII (GGCC), Alu I (AGCT)自制纳米
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•6 bp recognition sites: EcoRI (GAATTC), HindIII
(AAGCTT).
•Blunt ended cleavage: ScaI (AGT/ACT)
•Sticky ended cleavage
•5’ overhang: EcoRI (G/AATTC)
•3’ overhang: PstI (CTGCA/G)
•Electrophoresis of DNA fragments.
–Relationship between fragment size and mobility.•DNA and RNA hybridization.
–re-association of homologous single stranded nucleic acids to form double stranded nucleic acids, by
means of base complementarity
–highly sensitive method of detecting specific nucleic
acid sequences when one known nucleic acid is
labelled.
–can get hybridization between nucleic acids with less than 100% match (down to about 60-70%):
heteroduplexes
–Factors affecting hybridization:
•Nucleic acids: guanosine + cytosine content, homology,
length.
•Physical conditions: temperature, cation concentration
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(especially Mg++)
•Filter hybridization
–Capillary transfer of DNA fragments from agarose gels to membranes for “DNA gel blot
hybridization”(Southern blotting).
–Northern blotting: Transfer of RNA from gel to
membrane, hybridization with DNA probe.
–Western blotting: Transfer of proteins from gel to
membrane, probing with antibody.
–South-western blotting: Transfer of DNA-binding
proteins to membrane, probing with DNA.
–Preparation of bacterial colonies for colony
hybridization.
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显示–Preparation of bacteriophage plaques for plaquehybridization.
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