prepared by: nor helya iman kamaludin [email protected] ptt 202: organic chemistry for...

PREPARED BY: NOR HELYA IMAN KAMALUDIN [email protected] PTT 202: ORGANIC CHEMISTRY FOR BIOTECHNOLOGY

Nucleic acid molecules Ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) Both molecules are polymers of nucleotides. DNA is found in chromosomes, and genes are unique sequences of DNA nucleotides. The genes contain the inheritable information which together with RNA directs the synthesis of all the cells proteins. 3 components of RNA and DNA nucleotides Carbohydrate Phosphate group Organic nitrogenous base 5 common bases found in nucleic acids Purine bases : Adenine (A), Guanine (G) Pyrimidine bases: Cytosine (C), Thymine (T), Uracil (U) Location of bases in DNA and RNA Both DNA and RNA : A, G, C DNA only: T RNA only: U

Stage 1 The cells or tissue are broken open to release their contents. Stage 2 The cell extract is treated to remove all components except the DNA or RNA. Stage 3 The resulting DNA or RNA solution is concentrated. Stages in Isolation and Purification of Nucleic Acids

Types of Isolation Isolation of total cellular DNA Isolation of RNA

The tissue or cell is firstly homogenized in a buffer containing a detergent such as Triton X-100 and sodium deodecyl sulphate (SDS). This step is to disrupt the cell and dissociates DNA-protein complexes. Protein and RNA are then removed by sequential incubations with a proteolytic enzyme (usually proteinase K) and ribonuclease. Finally, the DNA is extracted into ethanol. Ethanol only precipitates long chain nucleic acid and thus leaves the single nucleotides from RNA digestion in the aqueous layer.

Alternative method for DNA isolation Using calcium chloride density gradient centrifugation Separates protein, RNA and DNA according to their buoyant densities.

Step 1 A density gradient is produced by centrifuging a solution of CsCl at high speed. Step 2 Macromolecules present in the CsCl solution during centrifugation will form bands at distinct points in the gradient depending on their bouyant density DNA has a bouyant density of 1.7 g/ml and will therefore migrate to this point in the gradient. Step 3 Protein has a much lower buoyant density will float at the top of the tube, while RNA will pellet at the bottom (Figure 1).

Figure 1; Caesium chloride density gradient centrifugation for (a) the separation of DNA from RNA and protein and (b) the separation of linear DNA and supercoiled DNA.

Problems encountered during RNA isolation The sample is easily contaminated by ribonuclease causing breakdown of the RNA. Endogenous ribonuclease activity is prevented by addition of inhibitors in the early stages while exogenous ribonuclease activity is minimized by using pure chemicals and sterile glassware. Tight association of RNA with protein as in polysomes. Harsh treatment are required to release the RNA from these complexes. 2 methods used for RNA isolation Proteinase K method Guanidine thiocyanate method

The cells are lysed by incubation in a hypotonic solution; leaves the nuclei intact. The cell debris and nuclei are pelleted by centrifugation leaving the cytoplasmic RNA free from DNA in the supernatant. The RNA is released from the polysomes by incubation with proteinase K. The protein extracted into phenol/chloroform solvent system. The RNA is then precipitated from the aqueous phase using ethanol. Proteinase K method

Step 1 The cells are lysed in a buffer containing strong chaotropic reagents such as guanidine thiocyanate and 2-mercaptoethanol. Important to denatures any ribonuclease present. Step 2 The supernatant is then placed on a cushion of CsCl (5.7 mol/l) and centrifuged at 100 000 g for 18 h. The RNA passes through the CsCl and is pelleted, while the DNA and protein remain in the aqueous solution. Step 3 The RNA pellet is dissolved in buffer and concentrated by precipitation in cold ethanol. Step 4 The mRNA is then isolated from this total cellular extract by affinity chromatography using oligo-dT-cellulose or poly(U)-sepharose. Guanidine thiocyanate method

Methods Spectrophotometric methods Chemical methods

Fundamental of analysis Relatively pure sample of DNA and RNA are used The presence of conjugated double-bond systems in the purine and pyrimidine bases means that DNA and RNA absorb light in the ultraviolet region at 260 nm. Quantitation method Approximate determination: assumed that a 50g/ml solution of double- stranded DNA (dsDNA) has an absorbance of 1 at 260 nm. Exact quantitation: obtained by comparing the ratio of the absorbance of the sample at 260 and 280 nm. Optical density (OD) is often used in place of absorbance. Pure DNA preparations should have an OD 260/OD 280 of 1.8. Ratio < 1.8: protein contamination > 1.8: presence of RNA

Determination of DNA concentration Bases that are not hydrogen bonded absorb more strongly than when base paired. Therefore treatment that disrupt the hydrogen bonding between the base pairs, such as heat or alkali, will increase the absorbance of DNA. When the solution of dsDNA is heated slowly, initially there is little change in the absorption until the melting temperature (T m ) is reached. Here, the hydrogen bonds are broken, producing a rapid increase in absorbance to a higher value, which is not significantly changed on further heating (Figure 2). In case where a small amounts of DNA to be detected by spectrophotometry, the fluorescent dye EtBr can be used to amplify the absorption.

Figure 2: Melting temperature for DNA

Application Used when there are large amounts of interfering substances present, such as tissue or cell homogenate. Burton method This is a spectrophotometric assay based on the reaction of diphenylamine with the deoxyribose moiety of DNA to produce a complex that absorbs at 600 nm. The reaction is specific for deoxyribose and RNA does not interfere. It can be used on relatively crude extracts where direct spectrometric determinations of DNA concentration are not possible.

DABA fluorescence assay Diaminobenzoic acid (DABA) reacts with aldehydes of the form RCH CHO to produce a strongly fluorescent compound. Acid-catalysed removal of the purine base from the nucleic acid exposes the 1 and 2 carbons of deoxyribose and produces such an aldehyde group. Deoxyribose is the predominant aldehyde present in mammalian cells and essentially the only one present in acid precipitates of aqueous extracts. Hence, no purification is required and RNA does not interfere. The method can be used on very small samples (e.g. 100 l)

What is DNA sequencing?? It is the sequence of the nucleotide bases in the DNA molecule that is fundamental to nucleic acid function. 2 methods The Maxam and Gillbert method The dideoxy method Description of methods Both depend on the production of a mixture of oligonucleotides labelled either radioactively or with fluorescein. This mixture of oligonucleotides is separated by high resolution electrophoresis on polyacrylamide gels and the position of the band determined. Known DNA sequence associated with specific genes are stored in computer libraries which are freely accessible. This allows the comparison of newly sequenced genes with those coding for known proteins.

Step 1 The single-stranded DNA fragment to be sequenced is end- labelled by treatment with alkaline phosphatase to remove the 5 phosphate. Step 2 Reaction with 32 P-labelled ATP in the presence of polynucleotide kinase, which attaches 32 P to the 5 terminal. Step 3 The labelled DNA fragment is then divided into four aliquots, each of which is treated with reagent which modifies a specific base. The four aliquots are shown in Figure 3.

Figure 3: Aliquots of DNA fragment

Step 4 After these reactions the four aliquots are incubated with piperidine, which cleaves the sugar-phosphate backbone of DNA next to the residue that has been modified. Step 5 The oligonuclectides in each aliquot are places in four different but adjacent wells of polyacrylamide gel containing SDS and separated by electrophoresis Step 6 The separated fragments are detected by autoradiography. The smallest fragments travel the furthest. Hence, the band that has travelled the furthest contains only one nucleotide, while the next band up the gel contains two nucleotides, etc. Step 7 The DNA sequence is therefore obtained by reading the gel from bottom to top (Figure 4).

Figure 4: The Maxam and Gillbert method of DNA sequencing.

Explanation of Figure 4 The labelled DNA strand is divided into four aliquots. Each is treated to cleave the strand next to a different base resulting in a mixture of different length nucleotides. These nucleotides are separated by polyacrylamide gel electrophoresis and the DNA sequence read from the gel

Step 1 Synthesis is carried out in the presence of the four deoxyribonucleotide triphosphates (ddNTPs), one of which is labelled with 32 P. Step 2 In the presence of competing ddNTPs, specific termination of the DNA synthesis occurs where the dideoxy derivative is incorporated instead of the deocyribonucleotide. Step 3 Four incubations are carried out, each in the presence of a different dideoxy derivative. Each incubation generates a heterogeneous population of labelled oligonucleotides terminating with the same nucleotide.

Step 5 Urea is added to each incubation to separate the two strands of DNA and the single strands are separated electrophoretically in adjacent lanes of an SDS polyacrylamide gel. Urea is also present in the gel to ensure that the strands of DNA stay separated. Step 6 The gel is then autoradiographed. The DNA sequence can be deduced from the ascending order of the bands in the for adjacent lanes.

Figure 5: The dideoxy (chain termination) method of DNA sequencing

Explanation of Figure 5 A complementary copy of the DNA strand is synthesized in the presence of dideoxy analoques of each base. Chain termination occurs where the analoque is inserted in place of the true base. The newly synthesized strands of DNA are separated by polyacrylamide gel electrophoresis and the sequence deduced from the pattern of bands.

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prepared by: nor helya iman kamaludin [email protected] ptt 202: organic chemistry for...

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