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Can two denatured DNA strands reassociate?

Can two denatured DNA strands reassociate?


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What matter if I increase the temperature to 95°C. So double-strand DNA denatured into two ssDNA, and then I decrease the temperature to room temperature. Are they able to reassociate after denaturation?


Yes, heating the DNA above 94°C for some length of time will melt, or denature, dsDNA into two ssDNA strands. Upon cooling, the complementary strands will come back to anneal. The degree and completeness of annealing, or association, is dependent on a number of factors including the rate of cooling and structural properties of the DNA itself (%GC content, secondary structure). I would not expect that the majority of ssDNA strands will completely anneal to a single, complementary dsDNA strand. What is more likely is that the DNA mix will look more like spaghetti with one strand partly annealed to two other strands. When performing PCR, the primers are designed to be specific, and added in excess, so that you bias the odds of annealing to favour a primer:ssDNA association. If this weren't the case, we could not possibly have efficient PCR amplification.


Can two denatured DNA strands reassociate? - Biology

Hybidization Technology

Renaturing Nucleic Acids

In contrast, the reannealing of two single strands is a bi-molecular reaction.

Two complementary single strands
must meet one another
and then complementary base pairs form between the two strands.

Because this is a bi-molecular reaction, the rate of the reaction depends on the concentration of the reactants - the two complementary strands.

Because reannealing is a bimolecular reaction, we are primarily interested in the rate at which the reaction occurs.

Reannealing reaction conditions are therefore chosen to MAXIMIZE the rate at which hybrids form.

The curve to the right shows how the rate of reannealing depends on temperature.

To maximize the rate of reannealing, we typically hybridize 15 o C below the Tm of the hybrid. Hybridization is also done at high salt to minimize the repulsion of the
sugar-phosphate backbones.

In order to visualize this concept, consider the reannealing of a variety of DNA samples.

To standardize conditions,
each DNA sample is sheared to random fragments 500 bp long.
Each DNA sample is at the same DNA (50 ug/ml) and NaCl concentration(1M).
Each sample is heated to boiling to denature it and then held at (Tm-15 o C) while the amount of DNA remaining single stranded is monitored.

The % single stranded is graphed as a function of

Cot - the initial DNA concentration (Co) times time (t)

Since the reaction follows bimolecular kinetics, we can obtain a value

Cot1/2 = Cot at which 1/2 of the DNA has reannealed

Cot1/2 measures the rate of reannealing which is inversely proportional to the concentration of complementary sequences being examined.

Small Cot1/2 values indicate that the complementary sequences are at high concentration.
(they reanneal very quickly, t is small)
Large Cot1/2 values indicate that the complementary sequences are at low concentration.
(they reanneal very slowly, t is large)

The following examples are ment to illustrate "Cot analysis (good luck)

The midpoint of the curve provides the value for Cot1/2 - the time required for half of the single strands to be in double stranded form at a defined initial DNA concentration.

In contrast to the above example, the E. coli genome consists of 5,000,000 bp of unique sequence.

Again, plotting the % single stranded as a function of Cot, we observe a single sigmoidal curve confirming that the E coli genome reanneals as a single kinetic class.

Notice that the Cot curve has shifted to the right relative to the lambda Cot curve. The increase in Cot1/2 reflects the number of copies of the E coli genome in solution as compared to the lambda genome. Since the E coli genome is 2 orders of magnitude larger than the lambda genome, any given 500 bp fragment will be present at 100 x lower concentration in the E coli genome sample.

This can be illustrated by considering what happens when the DNA sample consists of equal mass amounts of the lambda and E coli genomes (each at 25 ug/ml)

The Cot curve now has two kinetic components,
each comprising about 1/2 of the total DNA in the sample,
one with a Cot1/2 of the lambda genome,
one with a Cot1/2 of the E coli genome.

What about eukaryotes? What does the reannealing of eukaryotic genomes look like?

Unlike most prokaryotes, the Cot curves of eukaryotic genomes are complex curves. Modeling based on bimolecular kinetics allows us to 'fit' the data to define three kinetic classes that differ in their repetition frequency in the genome.

The first class represents a small portion of the genome (typically 10% or so) but is very highly repeated (10,000s of copies). These are short highly clustered repeated sequences found at eukaryotic telomeres and centromeres. Dispersed copies of these simple sequence repeats are also common.

The third kinetic class is the unique sequence of the genome. In this class we find most the the protein coding sequences of the genome. This class typically contains the majority of the sequence in the eukaryotic genome.


Examples of Denatured Proteins

Though protein denaturation is detrimental for cell survival, it is often encountered in daily life. For instance, egg white is primarily made of soluble proteins and is liquid and translucent in fresh eggs. When it is boiled, heat denatures the proteins and makes them lose solubility. Denatured proteins aggregate and form a mass that is now opaque and solid. Similarly, altering the pH of milk by adding acids such as citric acid from lemon juice denatures milk proteins and curdles the milk. The solid white portion that separates from the whey is denatured protein. This can also be seen when milk curdles naturally due to bacterial colonization. Bacteria can produce lactic acid as a byproduct of metabolism. When properly controlled, this process of denaturation is used to make yogurt and fresh cheese.


Kinetic classes of dna-biological discussion

In this article we will discuss about the repeated sequence of chromosomal DNA.

Eukaryotic genomes contain large amount of repetitive sequences, sometimes present in hundreds or thousands of copies per genome. The understanding of repetitive sequences is based on studies conducted on denaturation (separation of DNA double helix into its two component strands) and renaturation (re-association of the single strands into stable double-stranded DNA molecules) of DNA.

The two strands of a DNA molecule are held together by weak non-covalent bonds. When DNA is warmed in saline solution, a temperature is reached when two strands begin to separate, leading to single-stranded molecules in solution. This is called thermal denaturation or DNA melting.

The progression of thermal denaturation can be followed by observing increase in absorbance of the dissolved DNA. The nitrogenous bases of DNA absorb ultraviolet radiation with an absorbance maximum near 260 nm. In single stranded DNA, the hydrophobic interactions caused by base stacking are increased which increases the ability of the bases to absorb ultraviolet radiation.

The temperature at which the shift in absorbance is half completed is called the melting temperature (Tm) of DNA. The higher the GC content of the DNA, the higher the Tm. The reason being that there are 3 hydrogen bonds between G and C which confer stability on GC pairs, in comparison with AT pairs that are joined by two hydrogen bonds. Thus AT rich sections of DNA melt before the GC rich.

When denatured DNA is cooled slowly, the single strands reassociate to form double-stranded molecules, and properties of double helical DNA are restored, that is, it absorbs less ultraviolet light. This is called renaturation or reannealing. As described later, the property of reannealing has led to the development of methodology called nucleic acid hybridisation.

Britten and Kohne (1967) studied renaturation kinetics of DNA and discovered repeated sequences.

Walker (1969) distinguished 3 kinetic classes of DNA:

Fast reannealing fraction or highly repetitious DNA,

Intermediate reannealing fraction or moderately repetitious DNA, and

The slow annealing unique or single copy fraction.

1. Highly Repeated DNA Sequences:

Also called reiterated or redundant DNA. Consists of sequences present in at least a million copies per genome, constitutes about 10% of the total DNA in vertebrates. Such sequences are usually short, about a few hundred nucleotides long, and present in clusters in which the given sequence is repeated over and over again without interruption in tandem arrays (end-to-end manner). Highly repeated sequences include the satellite DNAs, minisatellite DNAs and the microsatellite DNAs.

Consists of short sequences about 5 to 100 bp in length. During density gradient centrifugation, satellite DNA separates into a distinct band, because the base composition of satellite DNA is different from that of bulk DNA. A species may have more than one satellite sequence as in Drosophila virilis which has 3 satellite sequences, each 7 nucleotides long.

Satellite DNA is present around centromeres in centromeric heterochromatin. In humans, 3 blocks of satellite DNA are present in the secondary constrictions of chromosomes 1, 9 and 16. A fourth block is present at the distal portion of the long arm of the Y chromosome.

These usually occur in clusters with about 3000 repeats, their size ranging from 12 to 100 bp in length. Minisatellite sequences occupy shorter stretches of the genome than the satellite sequences. Minisatellites are often unstable and the number of copies of minisatellites can increase or decrease from one generation to the next. The length of the minisatellite locus could vary within the same family, and in the population (polymorphism). Changes in minisatellite sequences can affect expression of nearby genes.


Materials and Methods

Plasmid/DNA treatments and denaturation/renaturation reactions

The genomic clones from the human DM locus (positions 357–433, as in 7) containing the (CTG)n·(CAG)n repeat, where n = 30, n = 50 and n = 255, have been described ( 4, 5, 8). The n = 30 and n = 50 repeat tracts are free of sequence interruptions, while the n = 255 tract contains four ACT interruptions resulting in a tract of (CTG)27ACT(CTG)40ACT(CTG)38ACT(CTG)40ACT(CTG)106±5. Plasmids were prepared using detergent lysis as previously described ( 5). Plasmids were treated with DNase-free RNases A and T1 (Sigma), phenol extracted, purified twice by isopycnic centrifugation and stored in TE (10 mM Tris, 1 mM EDTA, pH 7.6) at −20°C. All precautions were taken to avoid deletions during plasmid propagation in Escherichia coli ( 5, 9).

End-labeling of the 5′- or 3′-ends was performed using T4 polynucleotide kinase (USB) or AMV reverse transcriptase (USB) respectively as described ( 4, 5, 10, 11). All denaturation/renaturation reactions were performed as described and precautionary measures were taken to avoid sample dehydration ( 4, 5, 10, 11). All restriction enzymes were purchased from New England Biolabs and reactions were performed as specified by the manufacturer. The slipped-strand structures were excised from polyacrylamide gels and the DNA electroeluted as described ( 5, 10, 11).

Electrophoresis

Polyacrylamide gels (4%, 13 × 9 × 0.15 cm, 40:2 acrylamide: bis-acrylamide) were cast in TBE (90 mM Tris, 90 mM borate, 2.5 mM EDTA, pH 8.3) and subjected to a constant voltage (150 V, 10–12 V/cm) at room temperature, unless otherwise stated. Gels were then stained with ethidium bromide, photographed and/or dried on Whatman paper and exposed to radiographic film (Kodak) in the presence of an intensifying screen at −70°C or a PhosphorImager screen (Molecular Dynamics). The amount of S-DNA formed was measured by densitometric analyses as a percentage of the total population of repeat-containing molecules. All quantitation was done on 3–10 experiments by PhosphorImager analysis using ImageQuant software (Molecular Dynamics). All relative migrations and base pair estimations were calculated with respect to the migration of a 123 bp ladder (Gibco BRL) as previously described ( 8).

Electron microscopy

DNA samples were prepared and EM was performed as described ( 12). In brief, the samples were mixed with a buffer containing 0.15 M NaCl, 1 mM MgCl2 and 2 mM spermidine, adsorbed to glow-charged thin carbon films, washed with a water/graded ethanol series and rotary shadow cast with tungsten. Samples were examined using a Phillips CM12 electron microscope. Micrographs are shown in reverse contrast. A Nikon multiformat film scanner attached to a Macintosh computer and Adobe Photoshop was used to form montages of the images.

Denaturing and renaturing DM DNA fragments containing (CTG)n·(CAG)n repeats results in slowly migrating DNAs with respect to DNAs that have never been denatured/renatured. (A) Map of the DM (CTG)n·(CAG)nHindIII-EcoRI fragment. Human non-repetitive flanking sequences (positions 357–375 and 391–433, as in 7) are in bold. The thin lines represent plasmid vector sequences. (B) (CTG)n·(CAG)n-containing fragments with n = 30 and n = 50 are shown. Linear non-treated (LNT) controls (lanes 1 and 3) and reanealed (RD) (lanes 2 and 4) 32 P-labeled HindIII-EcoRI restriction digestion products were separated on a 4% polyacrylamide gel, dried and exposed for autoradiography. The brackets indicate the distribution of total radioactivity migrating anomalously following reannealing. The linear duplex DNAs are also indicated. The pattern of slow migrating products was indistinguishable when either the CTG or the CAG strands were radiolabeled, indicating that the new products were composed of both CTG- and CAG-containing strands. (C) Propensity of S-DNA formation as a function of repeat length. The percentages were measured as described in Materials and Methods.

Denaturing and renaturing DM DNA fragments containing (CTG)n·(CAG)n repeats results in slowly migrating DNAs with respect to DNAs that have never been denatured/renatured. (A) Map of the DM (CTG)n·(CAG)nHindIII-EcoRI fragment. Human non-repetitive flanking sequences (positions 357–375 and 391–433, as in 7) are in bold. The thin lines represent plasmid vector sequences. (B) (CTG)n·(CAG)n-containing fragments with n = 30 and n = 50 are shown. Linear non-treated (LNT) controls (lanes 1 and 3) and reanealed (RD) (lanes 2 and 4) 32 P-labeled HindIII-EcoRI restriction digestion products were separated on a 4% polyacrylamide gel, dried and exposed for autoradiography. The brackets indicate the distribution of total radioactivity migrating anomalously following reannealing. The linear duplex DNAs are also indicated. The pattern of slow migrating products was indistinguishable when either the CTG or the CAG strands were radiolabeled, indicating that the new products were composed of both CTG- and CAG-containing strands. (C) Propensity of S-DNA formation as a function of repeat length. The percentages were measured as described in Materials and Methods.


Repeated Sequence of Chromosomal DNA | Genetics

In this article we will discuss about the repeated sequence of chromosomal DNA.

Eukaryotic genomes contain large amount of repetitive sequences, sometimes present in hundreds or thousands of copies per genome. The understanding of repetitive sequences is based on studies conducted on denaturation (separation of DNA double helix into its two component strands) and renaturation (re-association of the single strands into stable double-stranded DNA molecules) of DNA.

The two strands of a DNA molecule are held together by weak non-covalent bonds. When DNA is warmed in saline solution, a temperature is reached when two strands begin to separate, leading to single-stranded molecules in solution. This is called thermal denaturation or DNA melting.

The progression of thermal denaturation can be followed by observing increase in absorbance of the dissolved DNA. The nitrogenous bases of DNA absorb ultraviolet radiation with an absorbance maximum near 260 nm. In single stranded DNA, the hydrophobic interactions caused by base stacking are increased which increases the ability of the bases to absorb ultraviolet radiation.

The temperature at which the shift in absorbance is half completed is called the melting temperature (Tm) of DNA. The higher the GC content of the DNA, the higher the Tm. The reason being that there are 3 hydrogen bonds between G and C which confer stability on GC pairs, in comparison with AT pairs that are joined by two hydrogen bonds. Thus AT rich sections of DNA melt before the GC rich.

When denatured DNA is cooled slowly, the single strands reassociate to form double-stranded molecules, and properties of double helical DNA are restored, that is, it absorbs less ultraviolet light. This is called renaturation or reannealing. As described later, the property of reannealing has led to the development of methodology called nucleic acid hybridisation.

Britten and Kohne (1967) studied renaturation kinetics of DNA and discovered repeated sequences.

Walker (1969) distinguished 3 kinetic classes of DNA:

Fast reannealing fraction or highly repetitious DNA,

Intermediate reannealing fraction or moderately repetitious DNA, and

The slow annealing unique or single copy fraction.

Kinetic Classes of DNA:

1. Highly Repeated DNA Sequences:

Also called reiterated or redundant DNA. Consists of sequences present in at least a million copies per genome, constitutes about 10% of the total DNA in vertebrates. Such sequences are usually short, about a few hundred nucleotides long, and present in clusters in which the given sequence is repeated over and over again without interruption in tandem arrays (end-to-end manner). Highly repeated sequences include the satellite DNAs, minisatellite DNAs and the microsatellite DNAs.

Consists of short sequences about 5 to 100 bp in length. During density gradient centrifugation, satellite DNA separates into a distinct band, because the base composition of satellite DNA is different from that of bulk DNA. A species may have more than one satellite sequence as in Drosophila virilis which has 3 satellite sequences, each 7 nucleotides long.

Satellite DNA is present around centromeres in centromeric heterochromatin. In humans, 3 blocks of satellite DNA are present in the secondary constrictions of chromosomes 1, 9 and 16. A fourth block is present at the distal portion of the long arm of the Y chromosome.

These usually occur in clusters with about 3000 repeats, their size ranging from 12 to 100 bp in length. Minisatellite sequences occupy shorter stretches of the genome than the satellite sequences. Minisatellites are often unstable and the number of copies of minisatellites can increase or decrease from one generation to the next. The length of the minisatellite locus could vary within the same family, and in the population (polymorphism). Changes in minisatellite sequences can affect expression of nearby genes.

These include the shortest sequences one to five base pairs long, present in clusters of about 50 to 100 base pairs in length. They are dispersed evenly throughout the DNA. The human genome contains about 30,000 different microsatellite loci. Changes in the number of copies of certain microsatellite sequences are responsible for some inherited diseases.

2. Moderately Repeated DNA Sequences:

These are partially redundant. The sequences are highly similar but may not be identical. This fraction includes sequences that are repeated within the genome from a few times to tens of thousands of times. The genes for RNAs and histones are of this type. They constitute 15% of the DNA in mouse, 45% in Xenopus, and 80% in wheat, onion and salmon.

3. Unique or Single-Copy Sequences:

These sequences are present only once in the genome, or at the most, in few copies. They have a slow rate of re-association. Most of the structural genes are found among the unique sequences. Mouse contains 70% and Xenopus about 55% of single copy sequences.

Dispersed Repeated Sequences:

Unlike repeated DNA described above in which repeated sequences are clustered in a tandem manner, there are some repeat sequences that are scattered throughout the genome, referred to as dispersed or interspersed DNA, instead of being clustered as tandem repeats. Dispersed repeated sequences have been studied in many organisms.

These are families of repeated sequences interspersed throughout the genome with unique sequence DNA. Often, small numbers of families have very high copy numbers and make up most of the dispersed repeated DNA in genome. In general, two interspersion patterns are encountered which allow these sequences to be classified as SINEs (short interspersed elements) or LINEs (long interspersed elements).

Families of SINEs have sequences about 100 to 400 bp long, whereas LINEs have about 1000 to 7000 bp . All eukaryotic organisms have LINEs and SINEs, although their relative proportions vary widely. Drosophila and birds have mostly LINEs, humans and frogs have mostly SINEs. LINEs and SINEs represent a significant proportion of all the moderately repetitive DNA in the genome.

Mammalian diploid genomes have about 500,000 copies of the LINE-1 (L1) family of repeated sequences representing about 15% of the genome. Other LINE families are much less abundant than LINE-1. Full length LINE-1 family members are 6 to 7 kilo bases long. The full length LINE-1 elements are transposons, that is, they encode enzymes for movement of these elements in the genome.

A good example of SINEs are the Alu sequences in mammalian genomes, so called because they contain a single site for the restriction endonuclease Alu I. Alu sequences are about 300 base pairs long, and about a million such sequences are dispersed throughout the genome, accounting for nearly 10% of the total cellular DNA.

Alu sequences are transcribed into RNA, but they do not encode proteins, and their function is not known. Significantly, like the LINE-1 sequences, Alu sequences are also transposable elements, and capable of moving to different sites in genomic DNA if enzymes required for movement are supplied by active LINE elements.

In Situ Localisation of Satellite DNA:

The precise locations of repeated DNA sequences on eukaryotic chromosomes have been determined by the technique of in situ hybridisation, first developed by Pardue and Gall (1970). The method is based on the fact that only those single strands of DNA/DNA or DNA/RNA hybridise which have complementary base sequences.

Cytological preparations of chromosome spreads are treated with NaOH which dissociates DNA. The preparations are incubated in a solution containing single-stranded nucleic acid molecules (either DNA or transcribed RNA), which are labelled with tritium. The regions of the chromosomes that contain complementary base sequences hybridise with the Corresponding sequences in single- stranded molecules. Their locations are determined by autoradiography.

Using labelled mouse satellite DNA, Pardue and Gall (1970) could determine the location of satellite sequences in the constitutive heterochromatin adjacent to the centromeres of mitotic chromosomes (Fig. 19.2c). Except for Y, all the remaining mouse chromosomes have satellite DNA at the centromeres. Later on many materials have shown satellite DNA in constitutive heterochromatin, that which forms C-bands with Giemsa.

Sometimes there may be more than one type of satellite DNA in a genome. Human chromosomes have 4 satellite sub-fractions present in chromosomes 1, 9, 16 and Y. All the 4 satellite sub-fractions hybridise with chromosome 9. It is also interesting from the evolutionary standpoint that all the human satellite sub-fractions hybridise with monkey and chimpanzee DNA.


Can two denatured DNA strands reassociate? - Biology

Hybidization Technology

Denaturing Nucleic Acids

In opposition to these stabilizing interactions is the electrostatic repulsion of
the charged sugar-phosphate backbone.

There are two basic approaches to denaturing double-stranded DNA
- heating and chemical treatiment.

Chemical denaturants can be divided into three classes

Before leaving the topic of DNA denaturation, lets look a little more closely at
Heat Denaturation.
Consider what happens when we heat a nucleic acid solution - say the E. coli genome. To prepare the DNA for this experiment, we shear it up into small pieces (approximately 500 bp long) and heat it slowly while monitoring the A260.

The initial A260 is stable until, over an interval of approximately 5 degrees C, the A260 suddenly increases by approximately 40%.

This increase in absorbance is referred to as the
Hyperchromic Shift.

The hyperchromic shift is due to the melting of the double helix into two single strands. The increased rotational freedom of the N-bases on strand separation accounts for the observed increase in absorbance.

The melting temperature , or Tm , is the temperature at the midpoint of the hyperchormic shift as shown to the left.

Three main factors affect the melting temperature.

The GC content of the nucleic acid sample.
This is due to the fact that AT base pairs share 2 H-bonds while GC base pairs share 3 H-bonds.

[salt]
Tm is sensitive to Na + concentration.
Na + acts to shield the negative charges of the sugar-phosophate backbone from interacting with one another. The repulsion between the negatively charged phosphate backbones is the major force destabilizing the double helix, therefore increasing Na + concentration increases helix stability and decreasing Na + concentration decreases helix stability.

DNA hybrid length
The longer the DNA hybrid is, the more H-bonds there are holding the two strands together. The longer the hybrid, the more H-bonds that must be simultaneously broken for the two strands to separate.
This is known as the 'zipper effect' after the (in)famous Canadian inventor Zippy. For our purposes we will only consider the two extremes of the zipper effect. For this course we will only consider the extemes of hybrid length - hybrids less than 50 bp (short) and those around 500 bp (long) in length.


Can two denatured DNA strands reassociate? - Biology

DNA denaturation, reannealing, hybridization are processes based on the chemistry of base pairing between complementary strands of DNA double helix.

The two strands of DNA double helix stay together via base pairing of nitrogenous bases: adenine, thymine, guanine, and cytosine. Remember, adenine (A) makes two hydrogen bonds with thymine (T), while guanine (G) forms three hydrogen bonds with cytosine (C). When these hydrogen bonds break, the double helix melts and generates two single strands. This process is called DNA denaturation (or DNA melting). High temperatures and certain chemicals induce denaturation of DNA. Because there are more hydrogen bonds between G and C base pairs than A and T base pairs, the more G-C base pairs a strand of DNA has, the higher the melting temperature.

Luckily, DNA denaturation is a reversible process. When the temperature is cooled down slowly, complementary single strands come together and make appropriate hydrogen bonds. As a result of this reannealing process, the two single strands recombine to form the double helix.

However, in a single-stranded state, DNA could bind to another single-stranded DNA of different origin if they have sufficient complementarity or enough base pairing. This is referred to as nucleic acid hybridization.

Practice Questions

Khan Academy

MCAT Official Prep (AAMC)

Section Bank C/P Section Passage 9 Question 70

Online Flashcards Biochemistry Question 5

Biology Question Pack, Vol 2. Passage 9 Question 58

• DNA denaturation is unwinding of the double helix at higher temperatures or extreme chemical conditions.

• When these conditions return back to favorable, DNA double helix forms again in a process of reannealing.

• DNA strands are not in all-out loyalty to each other: they can make hybridization, which is forming double strands with other complementary strands.

DNA denaturation:Refers to the melting of double-stranded DNA to generate two single strands.

Nitrogenous bases : Organic molecules, which are part of the nucleotides in DNA, showing base-like chemical properties.

Base pairing: The specific way in which bases of DNA line up and bond to one another A always with T and G always with C.

Double helix: The structure of DNA which looks like a twisted staircase.

Hybridization: The spontaneous pairing of complementary DNA sequence by hydrogen bonding to create a double-stranded molecule.


Sample

Experiment (B1-1). Properties of DNA
Background Information
DNA is an extremely long molecule that is very thin, yet quite rigid. The isolation of intact DNA molecules from a cell is difficult because of the relative ease with which these long rod-like molecules can be broken. Even the injection of a solution of DNA through the needle of a hypodermic syringe can cause extensive breakdown of DNA molecules. DNA can also be broken down by enzymes called deoxyribonucleases. Deoxyribonuclease I (DNAse I), an enzyme isolated from the mammalian pancreas, will be used in today’s experiment. This enzyme breaks the phosphodiester bonds that connect the nucleotide units in DNA, degrading long DNA molecules to a mixture of small nucleotide chains, as illustrated in Figure 1-1.

A DNA molecule is composed of two polynucleotide chains that are coiled around each other to form a rigid double-helix (see page 4). The double-helical structure of DNA is very stable at room temperatures because the hydrogen and hydrophobic bonds between the stacked bases hold the two polynucleotide chains together. However, if a solution of DNA is heated to a critical temperature, these bonds are broken and the two polynucleotide strands separate by a process called denaturation. DNA denaturation is accompanied by a decrease in the viscosity (thickness) of the solution because single-stranded DNA molecules form flexible coiled structures that no longer retain the rigid native structure of the DNA double-helix (Figure 1-2). If the DNA is cooled rapidly, the molecules will remain as single stranded polynucleotides. However, if the solution is cooled very slowly, restora¬tion of the DNA helix will occur. The reassembly of the two separated polynucleo¬tide strands is called renaturation.

Macromolecules such as DNA, RNA, and protein are not soluble in alcohol solutions, and precipitate (come out of solution) upon addition of alcohol. In general, globular proteins and RNA form fine, non-fibrous precipitates in alcohol. In contrast, the rod-like DNA molecules precipitate in alcohol as long fibers that can be spooled onto a glass rod. The ability of DNA to form fibers in alcohol depends on the physical properties of the DNA molecules. For example, DNA that has been broken into small pieces by DNAse I digestion will not form fibers, nor will single-stranded DNA that has been prepared by heat denaturation of the DNA double-helix. These properties of DNA will be illustrated in today’s experiments.


Study Questions and Analysis

1. Describe the basic properties of DNA that are responsible for fiber formation when alcohol is added to a solution that contains native (double-stranded) DNA.
2. Describe the type of precipitate that is formed when alcohol is added to denatured (single stranded) DNA. Offer an explanation as to why this precipi¬tate is different from that which is observed with native DNA.
3. Describe the action of DNAse Ion DNA and relate this effect to the results of your experiment in Section HI.


Summary

  • Reassociation kinetics defines different classes of DNA in genomes: single copy, middle repetitive and highly-repetitive
  • Reassociation kinetics makes it possible to determine the fraction of a genome in each kinetic class, and which transcripts are produced from each class

Authors: Dr. B. Fristensky and N. Brien

Unless otherwise cited or referenced, all content on this page is licensed under the Creative Commons License Attribution Share-Alike 2.5 Canada


Watch the video: Swedish In-Vitro Study - Spike Protein Goes to Nucleus and Impairs DNA-Repair (May 2022).


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