Labeling nucleic acids is primarily for use as probes. The probes are used to detect the presence, amount, or location of a particular nucleic acid by hybridization techniques like in situ hybridization, Northern blotting, and Southern blotting.
Nucleic acid labeling can also help determine synthesis or degradation rates.
Radioactive Atoms
Precursors used to label nucleic acids include radioactive isotopes like phosphorus-32 (32P), tritium (3H), and carbon-14 (14C).
Phosphorus-32 is preferred due to its stronger beta emission energy and short half-life (14 days), which facilitates waste disposal.
$^{32}P$ can be incorporated into NTPs or dNTPs at the alpha, beta, or gamma positions.
Only labeling at the alpha position allows incorporation of radioactivity into a nucleic acid chain during polymerization.
ATP labeled at the gamma position is useful for labeling with a kinase at the 5' end of a nucleic acid molecule.
Labeling of Total Nucleic Acids
Labeling total nucleic acids in living cells has various applications.
Deoxythymidine and uridine are specific precursors for labeling total DNA and RNA, respectively.
Cells are incubated with a labeled precursor for a short period (pulse) and then with an excess of non-labeled precursor (chase).
Extraction and quantification of the incorporated radioactivity allows for determination of total replication or transcription rates.
The determined rate is relative, requiring comparison between two experimental conditions.
Radioactive Probe Labeling
Any cloned DNA can be labeled in vitro and used as a probe.
Probe preparation involves in vitro incorporation of labeled nucleotides or molecules into nucleic acids.
Labeling can occur at one or both ends of nucleic acid molecules, creating low-density labeled probes.
High-density labeling is achieved by incorporating the labeled molecule uniformly throughout the nucleic acid molecule.
High-density labeled probes are preferred for hybridization experiments because they provide a stronger signal.
After the probe anneals with its target, its presence is detected by autoradiography.
Uniform Labeling
Nick-Translation
Nick-translation is an in vitro replication process where E. coli DNA polymerase I synthesizes new labeled DNA using appropriate precursors.
The reaction includes the template, enzyme, α-32P-dCTP (or another nucleotide), dNTPs, and a buffer.
Single-stranded nicks are randomly introduced in the dsDNA template using DNase-I.
DNA polymerase-I initiates DNA synthesis at the 3' end of the nicks, while its 5'-3' exonuclease activity excises nucleotides at the 5' end.
The position of the nicks is "translated" downstream, with labeled nucleotides replacing non-labeled ones.
The resulting probe is labeled on both strands and is highly radiolabeled, allowing it to hybridize to the DNA sequence of interest.
This method works best with linear DNA fragments larger than 500 bp.
Random Priming
A dsDNA is denatured, and DNA polymerase synthesizes newly labeled DNA through template-dependent extensions of random hexamer primers.
Random hexanucleotides contain all four bases in each position, making them suitable for use with any probe.
Polymerases lacking 5'-3' exonuclease activity, such as the Klenow fragment of E. coli DNA polymerase-I, are used.
In addition to the template and primers, α-32P-dCTP (or another nucleotide), dNTPs, and a buffer are added, and the mixture is incubated at 37°C for 15-30 minutes.
The template strand remains unlabeled, while the newly synthesized strand is completely labeled, resulting in a very high specific activity probe.
This method creates probes from 100 to 600 bp long and is particularly well-suited for preparing nonradioactive probes due to their high specific activity.
PCR Probes
Two techniques are used to prepare probes using PCR.
The first generates uniformly labeled probes by incorporating a labeled nucleotide during PCR.
The second synthesizes a large number of specific DNA target molecules by PCR, which are then used as a template for random-primer or nick-translated probes.
PCR labeling can be done using either genomic template without cloning or from a DNA fragment cloned into a plasmid.
Advantages of generating DNA probes by PCR include the ability to synthesize large amounts of probe with high label density from very little DNA, using purified or partially purified DNA as a template, and the flexibility as the preparation does not depend on RE site location.
It is also possible to prepare specific, single-stranded probes using a single primer.
5'-Labeling of Nucleic Acids
For some techniques, low-density labeling of probes (one radioactive atom per nucleic acid strand or molecule) is sufficient.
One such labeling is carried out by phosphatase/kinase, where phosphatase (BAP, CAP, SAP) removes the 5'-P.
T4 Polynucleotide Kinase, requiring γ-32P-ATP, catalyzes the transfer of the γ-phosphate group of ATP to a 5'-OH terminus.
Nucleic acid molecules labeled by this technique are used for electrophoretic mobility shift assays (EMSA), DNA footprinting, sequencing, and nuclease S1 protection.
Probe Purification
Before using a probe for hybridization, it is important to purify it to remove unincorporated radioactive nucleotides.
This is done by gel filtration chromatography using a Sephadex column, which consists of carbohydrate beads with pores that exclude nucleic acid molecules but allow free nucleotides to enter.
DNA molecules are excluded from the column and elute quickly, while unincorporated free nucleotides take longer to elute.
Further purification can be performed by phenol-chloroform extraction followed by ethanol-salt precipitation.
Non-Radioactive Labeling
Although radioactive labeling is widely used, fast, and convenient, it is expensive and risky.
Non-radioactive DNA labeling methods include nick translation and random primer labeling using special DNA precursor molecules like digoxigenin-dUTP (DIG-dUTP).
DIG-dUTP is incorporated during DNA synthesis and can be detected after hybridization using an anti-DIG-AP conjugate, which is an antibody that specifically reacts with DIG and is conjugated to alkaline phosphatase.
The location of the probe-target hybrid is visualized by colorimetric substrates that produce a precipitated colored product, or by a chemiluminescent substrate that produces light, detected by autoradiography.
RNA Probes
Plasmid vectors have MCS downstream from bacteriophage promoters (SP6, T7, or T3).
cDNA of interest can be inserted at the MCS between promoters SP6 and T7 or T3.
The cDNA can be transcribed in vitro into ss sense or anti-sense RNA from the linearized plasmid in the presence of radioactively labeled NTPs.
RNA probes can be easily purified from a DNA template by RNase-free DNase-I treatment.
RNA probes can produce stronger signals in hybridization reactions because they have high specific activity compared to ssDNA probes.
The yield of RNA probes is typically high because the template can be repeatedly transcribed.
Principle and Aim of Sequencing
Sequencing of a nucleic acid is the determination of its primary structure and is systematically carried out for every cloned DNA.
It is a powerful technique used in molecular biology, and advances in large-scale sequencing have allowed for the complete sequencing of many microbial genomes and completion of genome sequencing of many other organisms, including humans.
Knowing the complete genome sequence is changing biology and has brought about new scientific disciplines such as functional genomics and bioinformatics.
The basic principle of DNA sequencing involves generating DNA fragments of different sizes that terminate at a specific base and separating these fragments by gel electrophoresis.
Methods of Generating DNA Fragments
There are two methods that generate DNA molecules of different lengths in a base-specific manner.
The first method, introduced by Maxam and Gilbert (1980), is the chemical degradation method, which uses base-specific chemical cleavage of the DNA fragment; it is not detailed here.
The second method, by Sanger (1970), is the enzymatic or dideoxy-termination method and uses the enzymatic synthesis of DNA fragments.
Sanger (Enzymatic) Method
The enzymatic method of DNA sequencing utilizes properties of DNA polymerase to incorporate a 2'-3'-dideoxynucleotide (ddNTP) instead of deoxynucleotide into a growing DNA chain.
A ddNTP lacks a 3'OH group, which blocks further chain elongation.
DNA synthesis is initiated by DNA polymerase-I at only one end of the molecule where the oligonucleotide primer anneals to the template.
Thus, all synthesized DNA molecules will share an identical 5' end (the 5' end of the primer).
The 3' end will terminate at a specific base by incorporation of a ddNTP residue that will block DNA elongation.
Four separate primer extension reactions are initiated in different tubes using the same primer.
Each reaction contains all four usual dNTPs, but only one of the four ddNTPs.
Concentration of ddNTP in each of the four reactions is low so that incorporation occurs at random positions.
The ddNTPs are incorporated at random positions along the nucleotide chain, which causes random chain termination on the individual strands present in a solution.
This generates DNA fragments of different lengths, each terminated at the 3' end by a specific base.
If the primers are not labeled, a radioactively labeled nucleotide must be included in the synthesis, so that the labeled chains of various lengths can be visualized by autoradiography after separation by electrophoresis.
The enzymatic method has been improved for large-scale DNA sequencing projects, and a number of new modified enzymatic sequencing methods were developed, such as automated sequencing.
Electrophoresis and Autoradiography
Sequencing methods depend on electrophoresis in a high-resolution polyacrylamide gel to separate the fragments generated by the sequencing reactions.
The products in the four tubes are sorted in four distinct lanes (named A, T, C, and G) by electrophoresis on a denaturing polyacrylamide gel.
The gel is then dried, and autoradiography is performed by applying an X-ray film against the dried gel.
The sequence is read by switching among the four lanes and following the fragments by one nucleotide increase (or decrease). The 3' end is near the negative pole (the largest fragments), and the 5' end is near the front of migration (smaller fragments).
Automated Sequencing
The classical enzymatic technique was improved by the introduction of fluorescent labeling of nucleotides, which made automation of the procedure possible.
The primer is not labeled; rather, four distinct fluorochrome groups are independently coupled with the four ddNTPs.
The four enzymatic sequencing reactions are carried out in the same tube.
All the fragments have the same non-labeled 5' end (that of the primer) but differ in the 3' end where a labeled ddNTP is incorporated.
By the end of the reaction, tube content is sorted by electrophoresis on a polyacrylamide gel (on one single lane), and the bands are visualized after laser-induced fluorescence.
Since each fluorochrome type is associated with a specific ddNTP, each nucleotide type appears in a distinct color that corresponds to the fluorescing color.
Thus, the sequence is read according to band size and the emitted color.
Automated sequencing machines use the same principle of ddNTP-specific fluorochrome labeling and laser-induced fluorescence combined with the use of capillary electrophoresis instead of the usual gels.
Detection sensitivities at the output of the capillary are higher than classical radioactive bands (autoradiography) since laser-induced fluorescence of dyes is used, and the peaks are narrow.
RNA Sequencing
RNA can also be sequenced by the enzymatic and chemical methods using radioactive or fluorescent labeling.
The techniques are similar to the reactions with DNA templates, except for the requirements for reverse transcriptase in the enzymatic technique.
In the labeling reaction, a fluorescent or radioactive nucleotide is incorporated into the cDNA strands retrotranscribed from specific mRNA.
Specific primers can be designed based on DNA sequences or amino acid sequences of interest.
Genome Sequencing
The idea of sequencing the entire human genome was first conceived in the mid-1980s but was initially faced by broad skepticism among biologists.
In 1986, the project was launched and gained broader support in 1988.
Moreover, the project included sequencing the genomes of several model organisms and the parallel development of detailed genetic and physical maps of human chromosomes.
The first complete genome to be sequenced was that of the bacterium Haemophilus influenzae in 1995, and two draft sequences of the human genome were published in February 2001.
Different approaches were adopted to obtain the human genome sequence.
The first is the sequencing in a sequential manner of fragments derived from BAC (bacterial Artificial Chromosome) clones that had been previously mapped to human chromosomes; the second is the shotgun sequencing approach (sequencing of random fragments, and overlaps between fragments were then used to reassemble a complete genome sequencing).
Several important conclusions immediately emerged from human genome sequences.
First, the number of human genes appears to be between 30,000 and 40,000, substantially fewer than previous estimates of approximately 100,000.
Interestingly, however, alternative splicing appears to be common in the human genome, so each gene may encode an average of 3 different proteins.
Introns account for about 25% of the human genome, and repetitive sequences for about 60%.
It is noteworthy that over 40% of human DNA is composed of sequences derived by reverse transcription (retroposons), emphasizing the importance of this mode of information transfer in the evolution of our genome.
Sequence Databases (Information Superhighway): An Overview
Advances of recombinant DNA techniques produced an overwhelming flood of information.
The updated databases contain information about DNA, RNA, and protein sequences, as well as protein structure, genetic maps, and chemical formulas.
For each sequence submitted, a unique accession number will be given (e.g., M86389), which is the sequence name in the database.
The computer program used for this purpose is known as BLAST (basic local alignment search tool).
Principle of Hybridization
DNA-DNA and RNA-DNA hybridization reactions are the basis of many assays in DNA analysis and are presently some of the most frequently used techniques in molecular biology.
The hybridization reaction, using labeled probes, is the only practical way to detect the presence of specific nucleic acid sequences in a complex nucleic acid mixture.
Hybrid formation between complementary strands is commonly called a reassociation, renaturation, or reannealing reaction.
The reverse reaction is called strand separation, dissociation, denaturation, or melting of nucleic acids.
Hybridization in a liquid medium involves strands that are all soluble.
Hybridization on a solid support (e.g., nitrocellulose or nylon membrane, silicium or glass chips) involves strands that are soluble in the hybridization solution (named probe) and others fixed on the solid matter.
The hybridization reaction proceeds in two steps: the nucleation reaction and the "zippering" reaction.
The most frequently used hybridization technique is the membrane hybridization technique.
Southern Blotting
Principle and Aim
The principle of Southern blot combines resolving power of agarose gel electrophoresis in the separation of DNA fragments with the specificity of DNA-DNA or DNA-RNA hybridization reactions.
Details of the Technique
DNA extraction - digestion - electrophoresis
The experiment starts with DNA extraction followed by full digestion with specific REs.
The samples are loaded in the wells of an agarose gel whose concentration is chosen according to the size of fragments to be sorted (e.g., 1.5 to 2% gel for fragments less than 500bp and 0.8% gel for fragments about several kb).
Transfer
After electrophoresis, the gel may be stained with ethidium bromide to assess the quality of the samples.
DNA molecules are then transferred from the gel to a nitrocellulose or nylon membrane.
Transfer is indispensable since hybridization is impossible on the gel.
DNA transfer is generally accomplished by capillary methods, by electroblotting, and vacuum transfer procedures can also be used.
These other methods, in general, are faster than capillary transfer but are less efficient and require expensive equipment.
After transfer by any of the previous methods, nucleic acids must be fixed on the membrane in order to prevent the release of nucleic acid during hybridizations and washings.
This is performed by different means according to the type of membrane such as drying the membrane at 80°C (baking), alkaline treatment, and crosslinking by UV irradiation.
Hybridization
The membrane, which is a replica of the gel, is utilized for hybridization with a specific labeled probe.
First, pre-hybridization is performed using non-labeled non-specific DNA in order to saturate the membrane and avoid non-specific binding of the labeled probe.
Hybridization reaction requires adequate stringency (temperature depending on Tm and saline concentration).
Melting of DNA (or DNA/RNA ds) is independent of substrate concentration but depends only on nucleotide composition (GC %) and composition of the solvent (ionic strength).
To decrease hybridization temperature, the hybridization reactions are usually carried out in the presence of denaturing solvents while maintaining high ionic strength.
Prolonged hybridization at such high temperatures causes degradation of the probe and the target DNA (depurination).
Hybridization reaction is followed by washing to remove any unhybridized probe and melt mismatched hybrids.
Conditions of hybridization and washing that favor the formation and maintenance of high-fidelity hybrids are called high-stringency hybridizations (used to detect closely related sequences).
Conditions of hybridization that allow the formation of hybrids with many mismatched bases are called low-stringency hybridizations (used for detecting distantly related sequences and is frequently referred to as heterologous hybridization).
Autoradiography
Autoradiography of the membrane is performed by applying it against an X-ray photographic film which is impressed by the emitted rays thereby giving an image of radioactivity on the membrane.
Applications
There are many important applications of Southern blotting. In this chapter we emphasize its application in diagnosis (especially genetic diagnosis) and DNA fingerprints (RFLP).
Diagnosis
Southern blotting could be utilized for the diagnosis of defective genes (genetic diseases) as well as infectious diseases.
Another approach for genotype diagnosis consists to use the difference of restriction map between the normal allele and the mutant one (polymorphism of the restriction fragment length).
It happens that a mutation makes a gene defective without altering its restriction map.
DNA fingerprints (RFLP, RAPD)
All individual organisms, except clones and identical twins, have a unique genetic makeup although they have the same chromosome equipment.
Individuals can be genetically distinguished from one another using processes collectively described as DNA fingerprinting.
The two most common established methods for generating DNA fingerprints: the RFLP (restriction fragment length polymorphism) method of Jeffrey et al. (1985), and the RAPD (randomly amplified polymorphic DNA) technique developed by Williams et al. (1990).
Northern Blotting
Aim
Northern hybridization analysis determines the steady-state level of RNA transcripts.
Details of the Technique
The principle of Northern blot analysis is the same as that of Southern.
However, the detected molecule is an RNA instead of DNA.
It is however more difficult to perform than Southern largely because of degradation by RNases.
Total RNA molecules or total mRNA are extracted and sorted according to size by electrophoresis in a denaturing agarose gel.
Distinct samples are loaded in different wells.
It is important to load the same RNA amount from different samples in the wells of the gel in order to make their comparison possible.
The gel is stained with ethidium bromide in order to control the quantity and quality of the loaded samples by assessing the bands corresponding to the 28 and 18s ribosomal RNA.
Transfer and fixation of fractionated RNA samples is conducted similarly to Southern technique on a nylon or nitrocellulose membrane.
The membrane is prehybridized and then hybridized with a specific, denatured labeled probe.
After washing, probes hybridized to specific RNA types are visualized by autoradiography.
Membrane filters containing a record of different RNA species can be reused for analysis of multiple RNAs transcripts from several genes in the same RNA sample.
RT-PCR is an alternative of Northern
Analysis of mRNA expression by a semi-quantitative PCR approach is particularly useful when Northern blot cannot be applied because of low abundance of mRNAs, or when limited amounts are available.
Nuclease S1 (RNAse) protection assay is an alternative of northern
The principle of this assay is based on the ability of Nuclease S1 to digest single-stranded RNA.
Dot Blot (Slot Blot)
Dot blot share with northern blotting the aim of the determination of RNA relative amount.
However, it cannot determine the size of the revealed RNA since the molecules are not sorted by electrophoresis.
Instead, equal amounts of different samples are adsorbed and fixed as dots (about 3 mm in diameter) on a nitrocellulose or nylon membrane.
In Situ Hybridization
The previously described methods of nucleic acid detection are not able to determine the spatial distribution of the DNA or RNA of interest within the cells.
There are two commonly used procedures for in situ hybridization and detection of DNA or RNA; each has its own strengths and weaknesses.
One is radioactive in situ hybridization (RISH) using a radioactive probe which is usually a DNA molecule labeled by one of the previously explained methods.
The other procedure is fluorescence in situ hybridization (FISH) utilizing nonradioactive probes labeled with biotin or digoxigenin or a fluorescent dye.
Western Blotting
Principle
Western blotting is rather considered as a biochemistry technique and does not involve nucleic acid probes.
It is included in this chapter only because of its equivalence with Southern and Northern.
This technique answers the same questions as Northern but at the protein level.
Technique
Western blotting principle relies on the interaction between a specific antibody and its target protein on the membrane. Protein are sorted by electrophoresis
Proteins are extracted, and equal amounts from the samples are loaded in the wells.
During SDS-PAGE, proteins move through the gel matrix according to their molecular weights.
Transfer of proteins from a polyacrylamide gel to a nitrocellulose membrane achieved by electroblotting method (electrotransfer).
Determination of Transcription Initiation Site
After a gene is cloned, there is an important question to answer about the initiation site of transcription, which usually lies 20 to 30 bp downstream of the TATAA box.
The first approach to determining the transcription initiation site is known as protection against nuclease S1.
The other approach is named primer extension.
Principles and Aims of In Vitro Transcription (Run-On, Run-Off)
When gene expression is modulated (positively or negatively), the change can occur at the levels of RNA amount and/or protein amount and/or protein activity.
In vitro transcription is the synthesis of RNA in an acellular system. It aims either to produce RNA or to study the direct effect of certain agents on the transcription process.
There are two different methods of in vitro transcription: run-on and run-off.
Run-on aims to determine the transcription rate of any expressed gene.
In order to assess the transcription rate of a particular gene and compare it between two conditions or compare it to transcription rate of another gene, it is important to isolate and differentiate that specific RNA (after run-on) and determine the relative amount of incorporated radioactivity in that particular RNA
If we need to study transcription rate in two cell types or more, the described experiment is conducted in duplicate (or more) using different cell types
Investigation of RNA and Protein Stability
Even when transcription and translation rates of a particular gene are unchanged in the cell, there is still a possibility for RNA and protein amounts to be changed through modulation of their turn over (stability or half-life time).
Stability of RNA and proteins can be monitored by the techniques of Northern and Western blotting, respectively, provided that a specific protocol is applied.
After the start of treatment with actinomycin D, which is an RNA polymerase II inhibitor, cells are harvested at different times, and RNA are extracted, and Northern blotting is performed using a probe that is specific to the gene of interest.
Investigation of Translation Rate
In order to assess the translation rate in a cell, pulse-chase using a radioactive precursor of proteins is carried out.
In order to determine the translation rate of a particular protein, it is important to isolate that protein after pulse-chase by means of immunoprecipitation.
Foot Printing (Protection Against DNase I)
The aim of foot print is to determine, on a cloned DNA fragment, the sequences which are involved in interaction with proteins.
The principle of foot print is based on the fact that DNA becomes resistant to DNase-l in the sites that are occupied by, or interact with proteins.
Gel-Shift
EMSA detects and identifies binding of proteins to a single particular oligonucleotide (DNA and RNA).
Because of its high negative charge, an oligonucleotide (DNA or RNA) moves rapidly in polyacrylamide-gel electrophoresis.
If protein molecules are bound to the oligonucleotide, they will slow its movement through the gel; in general, the larger the bound protein, the greater the retardation of oligonucleotide migration.
EMSA can be combined with binding competition assay in order to identify the proteins in the complex.
Southwestern Blot
It is a method for detecting protein-DNA interactions by applying a labeled DNA probe containing the cis element to a transfer membrane (after sorting by electrophoresis) that contains renatured proteins.
DNA and Protein Arrays
Aim and Principle
Thousands of genes and their products (RNA and proteins) in a given living organism function in a complicated and orchestrated way that sustains cell and organism life through all development stages.
Traditional molecular biology techniques (Southern, Northern, and Western blotting) generally work on a "one gene in one experiment" basis.
In the late twentieth century, DNA microarray, a new high throughput technology, was developed and attracted tremendous interests among biologists.
Diversity of Arrays and Their Synthesis
An array is an orderly arrangement of samples (nucleic acid, proteins, antibodies, chemicals,…) that are adsorbed as tiny dots on a solid support made up of nylon (standard blotting membrane), glass, or silicium.
The nucleic acids extracted from cells are labeled and hybridized with the probe are named targets
An array is an orderly arrangement of samples (nucleic acid, proteins, antibodies, chemicals,…) which are adsorbed as tiny dots on a solid support made up of nylon (standard blotting membrane), glass or silicium (Table 10.1).
The content of each specific dot in the array is well known.
For instance, a nylon membrane where cloned cDNA corresponding to many genes are adsorbed as individual tiny dots at specific positions is an array.
Hybridization of probe with a mixture of labeled nucleic acid sequences (the targets) helps to identify them and/or to determine their relative amount.
Hybridization and Scanning
DNA microarrays allow rapid measurement and visualization of differential expression between genes at the whole genome scale.
Slide scanning is performed with a computer-assisted system.
Applications
Microarray-based hybridization analysis is a newly emerging technology with diverse applications in diverse fields such as gene discovery, functional genomic, disease diagnosis, and toxicological and pharmaceutical research
Genotyping. In the gene mapping application, microarrays are used to study DNA sequence polymorphisms, single-nucleotide polymorphisms (SNP), or small deletions/insertions in a specific chromosome
Protein Arrays
One of the most useful features of EMSA is the ability to monitor for DNA conformational changes due to protein binding.