Lecture 13 - Denaturation, Renaturation of Nucleic Acids PDF

Lecture 13 – Denaturation, renaturation of nucleic acids Chapter 6, 195-199, Chapter 7, 244-245 in Cox, Doudna, and O’Donnell: Molecular Biology (Principles and Practice), 2nd Edition (2015)  Definitions: Denaturation The process of separating the polynucleot...

Lecture 13 – Denaturation, renaturation of nucleic acids Chapter 6, 195-199, Chapter 7, 244-245 in Cox, Doudna, and O’Donnell: Molecular Biology (Principles and Practice), 2nd Edition (2015)  Definitions: Denaturation The process of separating the polynucleotide strands of duplex nucleic acid. Sometimes referred to as melting. Renaturation (also known as annealing:) restoration of typical base-pairing of two fully- separated complementary sequences, resulting in a native duplex structure. Melting temperature (Tm) (also known as annealing temperature): The temperature at which half of the nucleic acid helices are denatured Hybridization The process of forming a double helix from two complimentary single strands of nucleic acid (and be RNA and DNA, RNA and RNA and mixed oligonucleotides) doesn’t require 100% complementarity Fig. 6-28 2 Nucleic acid stability Helix random coil electrostatic repulsion of side chains (charge on phosphate group) higher ‘entropy’ of random coil Helix random coil hydrogen bonding between base pairs Base “stacking” (weak electrostatic van derWaals interactions) Fig. 6-29 3 Fig. 6-30 What factors affect double stranded nucleic acid stability? Base composition Biochemistry Tm depends on the G+C / A+T ratio, Tm increases with increased G+C content Voet & Voet 4e. Figure 5-17 (see figure at right) Length of base-paired nucleic acid sequence The length of the base-paired region of a nucleic acid duplex affects its thermal stability How many mismatches (unpaired bases) there are between strands Unpaired bases in the double helix lower its stability Salt concentration The higher the concentration of salt (cations) the higher the stability Other factors: pH, concentration of nucleic acids themselves, denaturing agents etc. 4 Calculating the Tm of double-stranded DNA Very accurate prediction of Tm requires enthalpy and entropy calculations – we won’t discuss this. However, a simplified general equation for DNA is as follows: @pH = 7 Glengan Tm = 64.9 + 41⨯([%G + C] − 16.4/L) at 50 mM NaCl with 50 nM oligonucleotide concentration at pH=7 Where %G+C is the percent G+C composition in the nucleic acid, and L is the length in nucleotides of the DNA For very short duplexes (14-20 base pairs), such as those formed by base-paired oligonucleotides, Tm can be calculated approximately with a very simple formula: Tm = 4°C (G + C)+ 2°C (A + T) Every 1% mismatch of bases in a DNA duplex reduces the Tm by ~1 °C. For an oligonucleotide of L=20, one mismatch is a 1/20 = 5% mismatch and lowers the Tm by 5 °C! 5 NOTE—We will not ask you to remember these equations for exams. IF you need them, we will provide them Calculating the Tm of double-stranded DNA Very accurate prediction of Tm requires enthalpy and entropy calculations – we won’t discuss this. However, a simplified general equation for DNA is as follows: Tm = 64.9 + 41⨯([%G + C] − 16.4/L) at 50 mM NaCl with 50 nM oligonucleotide concentration at pH=7 Where %G+C is the percent G+C composition in the nucleic acid, and L is the length in nucleotides of the DNA For very short duplexes (14-20 base pairs), such as those formed by base-paired oligonucleotides, Tm can be calculated approximately with a very simple formula: Tm = 4°C (G + C)+ 2°C (A + T) Every 1% mismatch of bases in a DNA duplex reduces the Tm by ~1 °C. For an oligonucleotide of L=20, one mismatch is a 1/20 = 5% mismatch and lowers the Tm by 5 °C! 6 NOTE—We will not ask you to remember these equations for exams. IF you need them, we will provide them Effect of temperature on rate of annealing (hybridization) The temperature affects the rate of nucleic acid hybridization. 25 below Renneling temp - - Im best Ser effects 100 I Self-hybridization [ Relative rate of 80 60 40 20 -55 -50 -45 -40 -35 -30 -25 -20 -15 -10 -5 -0 Temperature below Tm (°C) Maximum rate for DNA-DNA re-association occurs at ~25°C below Tm: the annealing temperature 7 Hybridization analysis Gene detection and mapping, gene expression studies, etc. Southern blotting DNA-DNA Northern blotting DNA-RNA Microarrays DNA-DNA Complex nucleic acid samples are fixed to solid surfaces (e.g., membranes, glass slides) Fixed samples are “probed” with a nucleic acid sequence of interest Hybridization kinetics are very similar to that of nucleic acids in solution that we’ve just discussed in the past few slides 8 In the next few slides we will discuss: How are nucleic acid probes synthesized and labeled? How are probe-target hybridizations detected? Hybridization analysis DNA labelling: Attachment of radioactive, fluorescent or other type of marker to DNA molecules. Numerous methods exist. For example: Random primer labelling: A labeling technique that uses a mixture of random oligonucleotides – usually hexamers (6-mers) – as primers to initiate DNA synthesis “Klenow fragment” of E. coli DNA pol I (the domain of the enzyme that has polymerase + 3’->5’ exonuclease activity) is used plus ”labelled” dNTPs Radioactive precursors are incorporated into DNA in a random fashion Russell,1992 Genetics (Box 15.1, Pg. 450) 10 Hybridization analysis Detection of labelled molecules Radioactive label: 35S 32P or 33P 35S 3H Labeled molecules detected with X-ray sensitive film (autoradiography) or a radiation-sensitive phosphorescent screen (phosphorimaging) Non-radioactive: Fluorescence—Molecules labeled with fluorophores (dyes) with different emission wavelengths, detected with film or fluorescence detector Chemiluminescence—Makes use of reaction between label and additional chemicals. Reaction generates light, signal detected with film e.g., Digoxigenin (DIG)-dUTP 11 Hybridization analysis Detection of labelled molecules Radioactive label: 35S 32P or 33P 35S 3H Labeled molecules detected with X-ray sensitive film (autoradiography) or a radiation-sensitive phosphorescent screen (phosphorimaging) Non-radioactive: Fluorescence—Molecules labeled with fluorophores (dyes) with different emission wavelengths, detected with film or fluorescence detector Chemiluminescence—Makes use of reaction between label and additional chemicals. Reaction generates light, signal detected with film e.g., Digoxigenin (DIG)-dUTP 12 Hybridization analysis 1 2 3 Southern blotting (Southern transfer / hybridization) Bad a probe (often restriction enzyme digested DNA run on agarose gel) 6 4 Named after Edwin Southern of Oxford University 5 of interest defect a gene Biochemistry Voet & Voet 4e. Figure 5-48 See also Fig 6-32 In Cox, Doudna, and O’Donnell 13 Hybridization analysis Northern blotting (Northern transfer / hybridization) An RNA extract is electrophoresed under denaturing conditions (e.g. formamide) in an agarose gel. After ethidium bromide staining, two bands are seen. These are the two largest ribosomal RNA (rRNA) molecules which are abundant in most cells. The smaller rRNAs, which are also abundant, are usually not seen because they are so short and they run off the bottom of the gel Sometimes you see a light “smear” of messenger RNAs (the transcripts of protein-coding genes) of various molecular weights (often barely visible compared to rRNAs). Large subunit rRNA Small subunit rRNA Light smear of mRNA 14  s Hybridization analysis detect which Northern blotting cellular tissues (Northern transfer / hybridization) contain the GOI An RNA extract is electrophoresed under denaturing conditions (e.g. formamide) in an agarose gel. After ethidium bromide staining, two bands are seen. These are the two largest ribosomal RNA (rRNA) molecules which are abundant in most cells. The smaller rRNAs, which are also abundant, are usually not seen because they are so short and they run off the bottom of the gel Sometimes you see a light “smear” of messenger RNAs (the transcripts of protein-coding genes) of various molecular weights (often barely visible compared to rRNAs). The gel is blotted on to a nylon membrane and, in this example, probed with a radioactively labeled DNA fragment. A single band is visible on the autoradiograph, showing that the DNA fragment used as the probe contains part or all of one expressed sequence of the mRNA Brown,1999 Genomes See also Fig 6-32 In Cox, Doudna, and O’Donnell (page 199) 15 Northern blot example Example: RNA extracted from different tissues probed with 32P labelled probe. Probe was cDNA for platelet endothelial cell adhesion molecule (PECAM-1) and GAPDH. Northern blot shows GAPDH mRNA is expressed in every tissue, but PECAM-1 (two isoforms) are mostly only expressed in kidney, lung and trachea Hybridization analysis detect the expression relative to of genes eachother ? Microarrays (microchip arrays, DNA chips, etc.) Glass surfaces (typically microscope slides) with thousands of DNA fragments arrayed at discrete sites (“spots”). The DNA spots are hybridized to complex samples of fluorescently labeled DNA or RNA in solution. Hybridization signals analyzed / compared. FIG. 7-31 A DNA microarray experiment. A microarray can be prepared from any known DNA sequence, from any source. Once the DNA is attached to a solid support, the microarray can be probed with other, fluorescently labeled nucleic acids. Here, mRNA samples are collected from frog cells at two different stages of development: single-cell stage (sample 1) and a later stage (sample 2). The cDNA probes are synthesized with nucleotides that fluoresce in different colors for each sample; a mixture of the cDNAs is used to probe the microarray. The probes anneal to spots containing complementary DNA; if the spot lights up, the corresponding gene is represented in the pool of mRNA used to produce the probes. Fig 7-31 (pg. 246) Green spots represent mRNAs more abundant at the single-cell stage; red spots, sequences more abundant later in development. The yellow spots indicate approximately equal abundance at both 17 stages.