Name: primer extension capture: targeted sequence retrieval from heavily degraded dna sources
Uploaded: 2009-09-03T11:00:00
Duration: 15 min 28 s
Description: Watch this Scientific Journal Video about Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources at JoVE.com

We thank M. Meyer for advice; The Croatian Academy of Sciences and Arts and the Berlin-Brandenburg Academy of Sciences for logistic and scientific support; and the Presidential Innovation Fund of the Max Planck Society for financial support. J.M.G. was supported by an NSF international postdoctoral fellowship (OISE-0754461). Sequences are deposited at the EBI nucleotide database with accession numbers: Neandertal 1 (Feldhofer 1) FM865407, Neandertal 2 (Feldhofer 2) FM865408, Sidron 1253 FM865409, Vindija 33.25 FM865410, Mezmaiskaya 1 FM865411

This method was used in the research reported in <a href="http://www.sciencemag.org/cgi/content/full/325/5938/318">Briggs et al., Science 325 (5938). 318-321 (2009)</a>.
Reagents required:
<ul>
 <li>AmpliTaq Gold DNA Polymerase </li>
 <li>10x GeneAmp PCR Buffer II </li>
 <li>MgCl2 25mM</li>
 <li>dNTP mix, 25 mM each</li>
 <li>BSA, 10 mg ml-1 in water </li>
 <li>Molecular biology-grade water</li>
 <li>EB buffer (supplied with MinElute PCR Purification kit); 10 mM Tris HCl, pH 8.5</li>
 <li>MinElute PCR Purification Kit</li>
 <li>M-270 streptavidin Dynabeads</li>
 <li>2x Binding and Wash (BW) buffer (2 M NaCl, 10 mM Tris-Cl, 1 mM EDTA, pH 8.0, 0.2% Tween 20)</li>
 <li>1x Binding and Wash (BW) buffer (2x BindWash buffer diluted 2X in water)</li>
 <li>Hot Wash (HW) buffer (2.5mM MgCl, 1X Taq Gold buffer, 0.1% Tween 20)</li>
</ul>
For manufacturer information regarding non-standard reagents and equipment see later table.
1) Library amplification
<ol>
<li>The DNA template here is a 454 library prepared from a low copy number DNA source as described in (Rohland and Hofreiter, 2007a, 2007b) and (Maricic and P&auml;&auml;bo, 2009). To amplify the whole library, prepare the following PCR mix:
<table border="1" cellspacing="0" cellpadding="0">
<tbody>
 <tr>
 <td>Reagent</td>
 <td>&mu;l per reaction</td>
 </tr>
 <tr>
 <td>Water</td>
 <td>45</td>
 </tr>
 <tr>
 <td>10X Gene Amp PCR buffer II</td>
 <td>10</td>
 </tr>
 <tr>
 <td>MgCl2 (25mM)</td>
 <td>10</td>
 </tr>
 <tr>
 <td>BSA (10 mg / ml) </td>
 <td>2</td>
 </tr>
 <tr>
 <td>dNTPs (25 mM each)</td>
 <td>1</td>
 </tr>
 <tr>
 <td>454 emPCR fwd primer/10uM</td>
 <td>3</td>
 </tr>
 <tr>
 <td>454 emPCR rvs primer/10uM</td>
 <td>3</td>
 </tr>
 <tr>
 <td>AmpliTaq Gold DNA polymerase (5 U / &mu;l)</td>
 <td>1</td>
 </tr>
 <tr>
 <td>454 DNA library template </td>
 <td>25</td>
 </tr>
 <tr>
 <td>Total</td>
 <td>100</td>
 </tr>
 </tbody>
</table>
</li>
<li>Use the following PCR program in a Thermo cycler for 14 cycles amplification
<ol>
<li>95&deg;C 12min</li>
<li>95&deg;C 30s</li> 
<li>60&deg;C (or desired annealing temp) 1 min</li>
<li>72&deg;C 1 min</li>
<li>Go to 2 (x 13)</li>
<li>72&deg;C 5min</li>
<li>10&deg;C &#x221E;</li>
</ol>
</li>
<li>Purify the reaction over a Qiagen MinElute spin column according to the manufacturer's instructions. Elute in 50 &mu;l buffer EB.</li>
<li>Quantify the purified amplified product as well as an aliquot of the unamplified library with qPCR (Meyer et al., 2007). If the amplified product has more than 1 X 1012 copies per ul, reduce the amount of template in the following Primer Extension step to ensure that not more than 2 X 1012 copies are added to the Primer Extension reaction.</li>
</ol>
2 ) Primer Extension
<ol>
<li>Prepare a master mix for the required number of reactions.
<table border="0" cellspacing="0" cellpadding="0">
<tbody>
 <tr>
 <td>Reagent</td>
 <td>&mu;l per reaction</td>
 </tr>
 <tr>
 <td>Water</td>
 <td>45</td>
 </tr>
 <tr>
 <td>10X Gene Amp PCR buffer II</td>
 <td>10</td>
 </tr>
 <tr>
 <td>MgCl2 (25mM)</td>
 <td>10</td>
 </tr>
 <tr>
 <td>BSA (10 mg / ml) </td>
 <td>2</td>
 </tr>
 <tr>
 <td>dNTPs (25 mM each)</td>
 <td>1</td>
 </tr>
 <tr>
 <td>454 emPCR fwd primer/10uM</td>
 <td>3</td>
 </tr>
 <tr>
 <td>454 emPCR rvs primer/10uM</td>
 <td>3</td>
 </tr>
 <tr>
 <td>AmpliTaq Gold DNA polymerase (5 U / &mu;l)</td>
 <td>1</td>
 </tr>
 <tr>
 <td>454 DNA library template </td>
 <td>25</td>
 </tr>
 <tr>
 <td>Total</td>
 <td>100</td>
 </tr>
 </tbody>
</table>
</li>
<li>Run the following program in a Thermocycler for the single primer extension reaction:
<ol>
<li>95&deg;C 12min</li>
<li>60&deg;C (or your PEC primer annealing temp if different) 1 min</li>
<li>72&deg;C 5 min</li>
<li>72&deg;C forever! 
CRITICAL STEP Keep reaction after extension at 72 &deg;C. Then directly pipette 150ul PBI or PB buffer for QIAGEN MinElute purification into the tubes before removing from the heat block. This is important to avoid non-specific primer annealing and capture as the mixture cools.
</li>
</ol>
</li>
<li>Purify the reaction over a Qiagen MinElute spin column, according to the manufacturer's instructions. Elute in 50 &mu;l EB.</li>
</ol>
3) Extension Product Capture
<ol>
<li>Resuspend stock solution of M-270 beads by vortexing. Take out 25 &mu;l bead suspension per sample. Wash the beads twice with 500ul 2x BW buffer and resuspend in 25 &mu;l 2xBW buffer per sample.</li>
<li>Add 25 &mu;l eluate from step 2.3 to 25 &mu;l bead suspension from step 3.1. Mix then rotate for 15 min at room temperature. 
 Recommended: keep remaining 5 ul of the eluate from step 2.3 for qPCR quantification. </li>
<li>Transfer the whole mixture to a fresh 1.5ml tube (this helps to reduce carryover of non-target library fragments). Pellet the supernatant using the Magnetic particle collector (MPC) and discard the supernatant.</li>
<li>Wash 5 times with 500 &mu;l 1xBW buffer (many washes help to remove as much background as possible). After the last washing step, spin down briefly and remove the last traces of supernatant.</li>
<li>Add 500&mu;l 1x Hot Wash (HW) buffer. Shake for 2 min at 65 &deg;C (or 5C above the PEC primer Tm) on a thermal block. Remove the supernatant quickly after this, to minimize cooling down. (This step is to remove background fragments still associated with the PEC primers but not actually extended on during the extension step). Remove the last traces of supernatant.</li>
<li>Resuspend the bead pellet in 30 &mu;l EB buffer. Transfer the whole mixture to a fresh 1.5ml tube (this helps to reduce carryover of non-target library fragments). Incubate at 95 &deg;C for 3 minutes in a thermal cycler for elution. Place in the MPC and remove the supernatant, taking care to leave the beads behind.</li>
</ol>
4) Capture Product Amplification
<ol>
<li>Prepare a PCR master mix for the required number of samples.
<table border="1" cellspacing="0" cellpadding="0">
<tbody>
 <tr>
 <td>Reagent</td>
 <td>&mu;l per reaction</td>
 </tr>
 <tr>
 <td>Water</td>
 <td>45</td>
 </tr>
 <tr>
 <td>10X Gene Amp PCR buffer II</td>
 <td>10</td>
 </tr>
 <tr>
 <td>MgCl2 (25mM)</td>
 <td>10</td>
 </tr>
 <tr>
 <td>BSA (10 mg / ml) </td>
 <td>2</td>
 </tr>
 <tr>
 <td>dNTPs (25 mM each)</td>
 <td>1</td>
 </tr>
 <tr>
 <td>454 emPCR fwd primer/10uM</td>
 <td>3</td>
 </tr>
 <tr>
 <td>454 emPCR rvs primer/10uM</td>
 <td>3</td>
 </tr>
 <tr>
 <td>AmpliTaq Gold DNA polymerase (5 U / &mu;l)</td>
 <td>1</td>
 </tr>
 <tr>
 <td>Eluted capture product </td>
 <td>25</td>
 </tr>
 <tr>
 <td>Total</td>
 <td>100</td>
 </tr>
</tbody>
</table>
</li>
<li>Use the following PCR program in a Thermo cycler for 14 cycles amplification:
<ol>
<li>95&deg;C 12min</li>
<li>95&deg;C 30s</li>
<li>60&deg;C (or desired annealing temp) 1 min</li>
<li>72&deg;C 1 min</li>
<li>Go to 2 (x 13)</li> 
<li>72&deg;C 5min</li>
<li>10&deg;C &#8734;</li>
</ol>
</li>
<li>Purify the reaction over a Qiagen MinElute silica spin column according to the manufacturer's instructions. Elute in 50 &mu;l EB buffer. The product can be now used either for a second round of capture, starting at step 2.1, or entered directly into the 454 emulsion PCR protocol, for sequencing.</li>
</ol>
Representative Results:
It is strongly recommended when starting out with the PEC protocol to perform first a capture reaction where a small amount of a 'positive control target sequence' (e.g. a ~100bp PCR product - 1 picogram is easily enough) is mixed in with the normal recommended amount of amplified 454 library template (see step 2.1), and capture is performed with a single PEC primer designed to capture the positive control product. If this control reaction is performed, qPCR with both positive control-specific and 454 adaptor-specific primer pairs can be used to quantify the amount of 'control target' versus background in the purified primer extension reaction (1.3), primer extension product (2.3) and eluted bead capture product (3.6). In this way, both the effectiveness of background removal ("specificity") and the efficiency of target recovery ("sensitivity") in your experimental conditions can be directly measured.
A successful PEC protocol normally has the following properties &#x2013;
Sensitivity (measured by amount of control target retained through the protocol): 
 Total amount of control target in eluted bead capture product (3.6) =~ 1-20% of total amount in purified primer extension reaction (2.3).
Background (measured by 454 emPCR primer pair): 
 Total amount of background in eluted bead capture product (3.6) &lt; 0.01% of total amount in purified primer extension reaction (2.3).
If there is less than 1% of the target remaining in the final product, the primer extension step may have been unsuccessful and should be investigated.
If there is much more than 0.01% of the background remaining in the final products, the washing steps may have been incorrectly performed and should be investigated.

<ol>
	<li>Briggs, A. W., Good, J. M., Green, R. E., Krause, J., Maricic, T., Stenzel, U., Lalueza-Fox, C., Rudan, P., Brajkovic, D., Kucan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+retrieval+and+analysis+of+five+Neandertal+mtDNA+genomes.">Targeted retrieval and analysis of five Neandertal mtDNA genomes.</a> Science. 325 (5938), 318-321 (2009).</li><li>Rohland, N., Hofreiter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+and+optimization+of+ancient+DNA+extraction.">Comparison and optimization of ancient DNA extraction.</a> Biotechniques. 42 (3), 343-352 (2007).</li><li>Rohland, N., Hofreiter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ancient+DNA+extraction+from+bones+and+teeth.">Ancient DNA extraction from bones and teeth.</a> Nat Protoc. 2 (7), 1756-1762 (2007).</li><li>Maricic, T., P&#228;&#228;bo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+454+sequencing+library+preparation+from+small+amounts+of+DNA+permits+sequence+determination+of+both+DNA+strands.">Optimization of 454 sequencing library preparation from small amounts of DNA permits sequence determination of both DNA strands.</a> Biotechniques. 46 (1), 51-57 (2009).</li><li>Meyer, M., AW, B. r. i. g. g. s., Maricic, T., Hober, B., Hoffner, B., Krause, J., Weihmann, A., Paabo, S., Hofreiter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+micrograms+to+picograms:+quantitative+PCR+reduces+the+material+demands+of+high-throughput+sequencing.">From micrograms to picograms: quantitative PCR reduces the material demands of high-throughput sequencing.</a> Nucleic Acids Res. 36 (1), e5-e5 (2007).</li></ol>

The authors have nothing to disclose.

The PEC method is simple, quick, sensitive and specific. Therefore we the authors envisage multiple applications outside ancient DNA, such as capture of small RNA fragments from an RNA library, interrogation of structural variation in a pooled sample or capture of 16S (or other loci) diversity from a metagenomic sample. One point to mention is that the sensitivity of capture becomes lower as the number of PEC primers in a multiplex capture reaction increases. PEC is therefore not ideally suited for capture of very large (e.g. a megabase or more) capture regions, but is extremely well suited for capture of small target regions or even SNP positions from many individuals in a rapid fashion.

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		<div>
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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>AmpliTaq Gold polymerase with GeneAmp PCR buffer II and MgCl2</td>
<td>&nbsp;</td>
<td>Applied Biosystems</td>
<td>N8080241</td>
<td>&nbsp;</td>
<tr>
<td>QIAGEN MinElute PCR purification kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28004</td>
<td>&nbsp;</td>
<tr>
<td>M-270 streptavidin beads</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>653.05</td>
<td>&nbsp;</td>
<tr>
<td>Tween-20</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P2287</td>
<td>&nbsp;</td>
<tr>
<td>DynaMag-2 magnetic particle separator</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
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primer extension capture: targeted sequence retrieval from heavily degraded dna sources

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ancient bones. The method greatly reduces sample destruction and sequencing demands relative to direct PCR or shotgun sequencing approaches. We used this method to reconstruct the complete mitochondrial DNA (mtDNA) genomes of five Neandertals from across their geographic range. The mtDNA genetic diversity of the late Neandertals was approximately three times lower than that of contemporary modern humans. Together with analyses of mtDNA protein evolution, these data suggest that the long-term effective population size of Neandertals was smaller than that of modern humans and extant great apes.

Watch this Scientific Journal Video about Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources at JoVE.com

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

We present a method of targeted ancient DNA sequence retrieval, which we used to reconstruct the complete mitochondrial genomes of five Neandertal individuals. Comparison of these sequences with present day humans suggests that Neandertals had a long term low effective population size.

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ...

primer-extension-capture-targeted-sequence-retrieval-from-heavily

Research

JoVE Journal

Biology

Retrieval of Mouse Oocytes

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus Genetic Stock Center (http://stkctr.biol.sc.edu), we are characterizing the salient differences needed to develop this system.  A primary goal has been to optimize oocyte/early embryo retrieval.

This protocol illustrates the technique for extracting oocytes or early-stage fertilized embryos from the oviduct of mice. The ability to identify the infindibulum and insert a blunt end needle into it is essential to correctly performing the procedure.

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology.  Together with the Peromyscus ...

Proboscis Extension Response (PER) Assay in Drosophila

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth (labellum), there are hair-like structures called sensilla which houses taste neurons. When an attractive substance makes contact to the labellum, the fly extends its proboscis to consume the material. Proboscis Extension Response (PER) assay measures this taste behavior response, and it is a useful method to learn about food preferences in a single fly. Solutions of various sugars, such as sucrose, glucose and fructose, are very attractive to the fly. The effect of aversive substances can also be tested as reduction of PER when mixed in a sweet solution.Despite the simplicity of the basic procedure, there are many things that can prevent it from working. One of the factors that requires attention is the fly's responsive state. The required starvation time to bring the fly to the proper responsive state varies drastically from 36 to 72 hours. We established a series of controls to evaluate the fly's state and which allows screening out of non-responsive or hyper-responsive individual animals. Another important factor is the impact level and the position of the contact to the labellum, which would be difficult to describe by words. This video presentation demonstrates all these together with several other improvements that would increase the reproducibility of this method.

Proboscis Extension Response PER Assay in Drosophila

Proboscis extension response or PER is a taste behavior assay that has been used in flies as well as in honeybees. When the proboscis makes contact with an attractive substance, the fly extends its proboscis to consume the substance. Solutions of various sugars are very attractive to the fly.

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth ...

Isolation of Genomic DNA from Mouse Tails

Primer-Free Aptamer Selection Using A Random DNA Library

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified through an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) to recognize a wide variety of targets, from small molecules to proteins and other macromolecules 2-4. Aptamers have properties that are well suited for in vivo diagnostic and/or therapeutic applications: Besides good specificity and affinity, they are easily synthesized, survive more rigorous processing conditions, they are poorly immunogenic, and their relatively small size can result in facile penetration of tissues.
Aptamers that are identified through the standard SELEX process usually comprise ~80 nucleotides (nt), since they are typically selected from nucleic acid libraries with ~40 nt long randomized regions plus fixed primer sites of ~20 nt on each side. The fixed primer sequences thus can comprise nearly ~50% of the library sequences, and therefore may positively or negatively compromise identification of aptamers in the selection process 3, although bioinformatics approaches suggest that the fixed sequences do not contribute significantly to aptamer structure after selection 5. To address these potential problems, primer sequences have been blocked by complementary oligonucleotides or switched to different sequences midway during the rounds of SELEX 6, or they have been trimmed to 6-9 nt 7, 8. Wen and Gray 9 designed a primer-free genomic SELEX method, in which the primer sequences were completely removed from the library before selection and were then regenerated to allow amplification of the selected genomic fragments. However, to employ the technique, a unique genomic library has to be constructed, which possesses limited diversity, and regeneration after rounds of selection relies on a linear reamplification step. Alternatively, efforts to circumvent problems caused by fixed primer sequences using high efficiency partitioning are met with problems regarding PCR amplification 10.
We have developed a primer-free (PF) selection method that significantly simplifies SELEX procedures and effectively eliminates primer-interference problems 11, 12. The protocols work in a straightforward manner. The central random region of the library is purified without extraneous flanking sequences and is bound to a suitable target (for example to a purified protein or complex mixtures such as cell lines). Then the bound sequences are obtained, reunited with flanking sequences, and re-amplified to generate selected sub-libraries. As an example, here we selected aptamers to S100B, a protein marker for melanoma. Binding assays showed Kd s in the 10-7 - 10-8 M range after a few rounds of selection, and we demonstrate that the aptamers function effectively in a sandwich binding format.

SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions. These fixed primer sequences can interfere with the selection process (false positives and negatives). Here we present a primer-free protocol.

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is achieved by reverse transcription of the RNA of interest using specific fluorescently labeled primers and subsequent analysis of the resulting cDNA fragments by denaturing polyacrylamide gel electrophoresis. Simultaneously, a traditional Sanger sequencing reaction is run on the gel to map the ends of the cDNA fragments to their exact corresponding bases. In contrast to 5&#39;-RACE (Rapid Amplification of cDNA Ends), where the product must be cloned and multiple candidates sequenced, the bulk of cDNA fragments generated by primer extension can be simultaneously detected in one gel run. In addition, the whole procedure (from reverse transcription to final analysis of the results) can be completed in one working day. By using fluorescently labeled primers, the use of hazardous radioactive isotope labeled reagents can be avoided and processing times are reduced as products can be detected during the electrophoresis procedure.
In the following protocol, we describe an in vivo fluorescent primer extension method to reliably and rapidly detect the 5&#39; ends of RNAs to deduce transcriptional starting points and RNA processing sites (e.g., by toxin-antitoxin system components) in S. aureus, E. coli and other bacteria.

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires precise identification of cells subpopulations from a heterogeneous tissue. Identification of cells of interest for LCM is usually based on morphological criteria or on fluorescent protein reporters. The combination of LCM and rapid immunolabeling offers an alternative and efficient means to visualize specific cell types and to isolate them from surrounding tissue. High-quality RNA can then be extracted from a pure cell population and further processed for downstream applications, including RNA-sequencing, microarray or qRT-PCR. This approach has been previously performed and briefly described in few publications. The goal of this article is to illustrate how to perform rapid immunolabeling of a cell population while keeping RNA integrity, and how to isolate these specific cells using LCM. Herein, we illustrated this multi-step procedure by immunolabeling and capturing dopaminergic cells in brain tissue from one-day-old mice. We highlight key critical steps that deserve special consideration. This protocol can be adapted to a variety of tissues and cells of interest. Researchers from different fields will likely benefit from the demonstration of this approach.

Gene expression analysis of a subset of cells in a specific tissue represents a major challenge. This article describes how to isolate high-quality total RNA from a specific cell population by combining a rapid immunolabeling method with laser capture microdissection.

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires ...