BIOC0005 Molecular Biology Lectures 3 & 4 2024 PDF

BIOC0005: Molecular Biology Lecture 3: Overview of molecular techniques and tools (cont.) Saul Purton  DNA cleavage using restriction enzymes Type II restriction enzymes: Bacterial enzymes that recognize and cut a specific p...

BIOC0005: Molecular Biology Lecture 3: Overview of molecular techniques and tools (cont.) Saul Purton  DNA cleavage using restriction enzymes Type II restriction enzymes: Bacterial enzymes that recognize and cut a specific palindromic sequence Enzymes are homodimers Recognise 4-8 bp sequences The sugar-phosphate backbones are broken leaving a 5’phosphate Depending on the enzyme, cleavage can leave a 3’ overhang, 5’ overhang (so-called sticky ends) or a blunt end Over 400 type II REs discovered Used for cloning, mapping, allele analysis (RFLP), etc. Fig from: https://www.sciencelearn.org.nz/resources/2035-restriction-enzymes  Cutting and joining DNA (“cloning”) Two DNA molecules can be joined if the ends are compatible. So can, for example, clone DNA into a plasmid. https://www.addgene.org/protocols/dna-ligation/  Generating restriction maps Using different combinations of REs allows us to map their approximate positions on a DNA molecule Alternatively, if we know the DNA sequence, we can use software to search for the RE sequences and generate a restriction map. Restriction Fragment Length Polymorphism (RFLP) analysis https://www.news-medical.net/life-sciences/Restriction-Fragment-Length-Polymorphism-(RFLP)-Technique.aspx  Other key enzymes using in molecular biology 1: DNA ligase DNA ligases catalyse the formation of a phosphodiester bond in double-stranded DNA. Bond formed between 5’phosphate and 3’ OH of adjacent nucleotides. Require an energy source (ATP or NAD+). Blunt end ligations much less efficient. 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ https://uvmgg.fandom.com/wiki/DNA_Ligase  2: DNA phosphatase Alkaline phosphatases such as calf intestinal phosphatase remove 5’-phosphate from DNA or RNA Used to prevent self-ligation of vector DNA P OH HO P CIP HO OH P OH HO OH HO P Ligation still possible X Cannot ligate to itself  3: DNA polynucleotide kinase PNK adds 5’-phosphate from ATP onto 5’ end of double-stranded or single-stranded DNA Used to label DNA with 32P 5’ 5’ HO OH HO OH HO OH 5’ PNK + ATP PNK + ATP P OH P OH HO P or allow ligation (e.g. of PCR products) HO OH HO OH  4. Polymerases Polymerases make nucleic acid polymers Use nucleotides (deoxyribonucleotides or ribonucleotides) as building blocks, and an existing nucleic acid strand (DNA or RNA) as a template for copying Four classes: Newly synthesised strand is DNA Newly synthesised strand is RNA DNA dependent DNA polymerase DNA dependent RNA polymerase DNA used as template (= DNA polymerases) (= RNA polymerases) RNA dependent DNA polymerase RNA dependent RNA polymerase RNA used as template (= Reverse transcriptases) (= RNA replicases) Are also other types of DNA or RNA polymerases that extend an existing strand. e.g. Terminal deoxynucleotidyl transferase (TdT) [DNA dependent DNA polymerase] Figure from DOI:10.1016/j.bdr.2015.02.005 poly(A) polymerase [RNA dependent RNA polymerase] lack any intrinsic specificity for their DNA or RNA substrate  DNA polymerases First one used in molecular biology was E. coli DNA polymerase I Klenow fragment Intact DNA Pol I Single polypeptide of 928 residues (~103 kDa) Has three separate activities: 5’–>3’ polymerase activity 3’–>5’ proof-reading activity 5’–>3’ exonuclease activity (RNA primer removal on lagging strand) Third activity unsuitable for many applications, but can be enzymatically cleaved off by the protease subtilisin. https://en.wikipedia.org/wiki/Klenow_fragment#/media/File:PolymeraseDomains.jpg = ‘Klenow fragment’ Klenow used for: Synthesis of dsDNA from single-stranded templates (e.g. in Sanger sequencing) Filling in recessed 3' ends of DNA fragments to make blunt ends Digesting away protruding 3' overhangs Preparation of radioactive DNA probes  Thermostable DNA polymerases (1) PCR method originally used Klenow as the DNA polymerase (see Saiki et al. 1985 on Moodle) Fresh enzyme was added every cycle since heating killed the polymerase Major advance was discovery of thermostable polymerase – Taq Polymerase – isolated from thermophilic bacterium, Thermus aquaticus Optimum activity is 75–80 °C, with a half-life of >40 mins at 95 °C Similar structure to DNA pol I, but proof-reading domain not functional, and 5’-3’ exonuclease activity does not degrade DNA Structure of Taq polymerase DOI: 10.3389/fmicb.2014.00565 primers. Recombinant protein now produced in E. coli for PCR market  Thermostable DNA polymerases (2) Although Taq polymerase is a reliable and relatively cheap enzyme for PCR, it has several drawbacks: 1. No proof-reading activity, therefore higher error rates during strand synthesis compared to (e.g.) DNA pol I 3’ 2. Lack of proof-reading typically results in addition of 3’ A overhangs of a single ‘A’ to PCR products A 3’ PCR product 3. Thermotolerance poor at the very high temperatures needed to denature GC-rich DNA So, companies have exploited DNA polymerases from hyperthermophilic bacteria and archaea, with the latter generally having higher fidelity owing to ‘proof-reading’ (3’–5’ exonuclease activity)  Thermostable DNA polymerases (3) Pfu Polymerase from hyperthermophilic archaeon Pyrococcus furiosus Two-subunit enzyme. High thermo-tolerance and proof-reading activity (error-rate ~ten-fold lower than Taq polymerase), but slower synthesis rate than Taq polymerase Phusion Polymerase A novel Pyrococcus-like enzyme fused with a processivity-enhancing domain. Shows increased fidelity (52x more accurate than Taq, 6x more accurate than Pfu) and high synthesis rate (1 kb every 15–30 seconds) https://www.neb.com/products/pcr-qpcr-and-amplification-technologies/phusion-high-fidelity-dna-polymerases/phusion-high-fidelity-dna-polymerases  Terminal deoxynucleotidyl transferase (TdT) Eukaryotic enzyme that adds random bases to 3’ end of single- or double-stranded DNA Does not require a template to copy, just a 3’–OH group Used for: homopolymer tailing labeling of the 3′ ends of DNA with modified nucleotides (e.g., ddNTP, DIG-dUTP) Being explored as an enzyme for ‘biological’ oligonucleotide synthesis  RNA polymerases (RNAP) Eubacterial-type (e.g. E. coli RNAP) Multiple subunit complex (22 core) with sigma factor for promoter recognition Uses regulated transcription in vivo from lac promoter Transcription in vitro from E. coli promoters Bacteriophage-type (e.g. T7, T3 & SP6 RNAP) Single subunit enzyme with both promoter recognition and RNA synthesis activities High specificity for promoter and high fidelity Uses regulated transcription in vivo from phage promoter Transcription in vitro  Reverse transcriptases (RT) RNA-directed DNA polymerases from retroviruses HIV RT Use RNA (or can use SS DNA) as a template to make a complementary DNA (Wikipedia) (= cDNA) strand Like all DNA polymerases, they require a 3’–OH group to extend an existing strand (here, this is provided by an annealed primer), rather than RNA polymerases which start strand synthesis de novo Two RT’s commonly used in molecular biology: Avian Myeloblastosis Virus (AMV) Reverse Transcriptase Moloney Murine Leukaemia Virus (MMLV) Reverse Transcriptase Uses: cDNA synthesis RT-PCR https://microbeonline.com/rt-pcr-principles-applications/ Considerations when choosing an RT: Fidelity, thermostability; processivity Key take-home points The precise and predictable action of enzymes that cut DNA or modify DNA ends can be used to clone or label DNA pieces. Polymerases are typically ‘copying’ enzymes and can be classified depending on type of template used and type of strand synthesized Are also other polymerases that add to the 3’ end of a strand Recombinant forms of enzymes used in a range of different molecular techniques. Key considerations are fidelity, thermostability, catalytic rate, etc.