BioProtocols for Nucleic Acids (DNA-1)

DNA (In Alphabeta)

ABC Random Amplification
Alcohol Precipitation of DNA
AML sample DNA preparation

Agarose gel electrophoresis

Agarose gel electrophoresis

Boomerang DNA Amplification Single primer DNA amplification

cDNA amplification from l-phage library

cDNA library screening

Characterization of DNA

Collection of Materials for DNA Analysis

Colony Hybridization

Colony Hybridization Protocols

Deoxyribose Isolation from DNA Degrasion

DNA Isolation, Purification and Troubleshooting

DNA Labeling Protocols

DNA ligation Protocol

DNA Methods （3）

DIG DNA labelling and hybrid detection (Boehringer Mannheim)

Direct Automated Sequencing off Bacterial Genomic DNABruce Rhoe

DNA extraction from agarose (Qiagen)

DNA - dische diphenylamine determination

DNA Fragment Isolation from LM agarose

DNA Ligation

DNA detection
DAPI chromosome identification

DNA preparation by cryostom tissue dissection
DNA labeling by nick translation

Ethanol precipitation of DNA

Extraction of DNA from bovine spleen

Fluorometry

BioProtocols for Nucleic Acids (DNA-2)

FISH

Genome DNA Extraction

Genomic DNA Isolation from Specific Samples

Genomic DNA preparation (plants)
Hybridization

In-Situ hybridization to Embryonic Tissue Section

Large Scale Plasmid Preparation: MGH

Methylene Blue DNA staining protocol

Microsatellites Protocols

Mitochondrial DNA Isolation

Modifications of DNA ends

PCR amplification of DNA

PCR analysis of DNA from Laser Microdissected (LM) Samples - Embryo Genotyping Protocol

PCR product purification (Qiagen)

Perfectly Blunt Cloning of DNA (Novagen)

Phenol/chloroform extraction of DNA

Plasmid midi-prep from bacteria

Plasmid Protocol--(1-10)

Plasmid Protocols--(21-30)

Plasmid Protocols--(31-37)

Preparation of genomic DNA from bacteria

Purification of DNA

Q-PCR

Random Primed labeling of DNA (Prime-It II -Stratagene)

Restriction enzyme digestion of DNA
Southern Hybridization
Stark's Prehybridization Buffer

Sequencing using Sequenase

Southern blot

Tail DNA prep

Wizard Plus Mini-preps (109. Plasmid Mini-prep)

Worm Genomic DNA: MGH

Worm genomic Southern blots: Michael Koelle

BioProtocols for Nucleic Acids (RNA)

RNA (In Alphabeta)

Amino-allyl labeling
BioAnalyzer

C. elegans RNA prep: Michael Koelle

IN VITRO TRANSCRIPTION OF RNA

In vitro transcription reaction

Isolation of poly-A RNA

LiCl RNA Preparation: MGH

Northern blots

Northern Blots: Michael Koelle

Nucleic Acids Research Methods (2)

Nucleotide composition of RNA

Orcinol determination of RNA

Phenol extraction of rRNA (rat liver)

PROTOCOL FOR SINGLE CELL mRNA AMPLIFICATION (REVERSE NORTHERN ANALYSIS)

Protocol: Fluorescence in situ hybridization

Protocols for SiRNA

Reverse Transfection Protocols

RNA amplification
RNA gel electrophoresis

RNA and protein extraction from the same sample

RNA Isolation

RNA MicroArray Protocol

RNA prepararion (from plants tissue)

RNA Preparation: MGH

RNEasy Midi

SELEX—in vitro selection

shRNA Protocols

Spectrophotometric analysis of RNA

Sucrose density fractionation

TRIzol plus RNEasy Mini
TRIzol product insert

Worm RNA Preparation: MGH

Nucleic Acids Research Articles and Protocols

Cell biology (8 Articles)

Chromatin (4 Articles)

Cloning (35 Articles)

Computational methods (92 Articles)

DNA characterisation (96 Articles)

DNA transfer (30 Articles)

Enzyme assays (24 Articles)

Genomics (74 Articles)

Microarray (48 Articles)

Miscellaneous/other (62 Articles)

Monitoring gene expression (63 Articles)

Mutagenesis (42 Articles)

New Restriction Enzymes (17 Articles)

Nucleic acid amplification (66 Articles)

Nucleic acid modification (42 Articles)

Nucleic acid structure (20 Articles)

Polymorphism/mutation detection (112 Articles)

Protein-nucleic acid interaction (50 Articles)

Protein-protein interaction (16 Articles)

Recombinant DNA expression (24 Articles)

Recombination (54 Articles)

Repair (20 Articles)

Replication (13 Articles)

RNA characterisation and manipulation (59 Articles)

Targeted inhibition of gene function (14 Articles)

HHV-6 is Passed to Children Through DNA

Adrienne Dellwo, Fibromyalgia & CFS Blog

ABSTRACT

NEWSBRIEF: A virus suspected of triggering some cases of chronic fatigue syndrome (CFS or ME/CFS) can be passed genetically from parent to child, according to an article published in the journal Pediatrics.

Experts used to believe that mothers passed human herpesvirus 6 (HHV-6) to their babies through blood exchanged during childbirth. HHV-6 causes roseola in infected children. New evidence shows the virus can come from either parent in the genetic material.

Nearly everyone is infected with HHV-6 at some point in their lives, but researchers are now investigating what it means to have a virus integrated into your genetic matter -- whether it can activate and cause problems, or cause an immune response just by being there.

Guide note: Could this be causing a genetic predisposition to chronic fatigue syndrome or autoimmune diseases? If so, genetic testing may someday be able to diagnose the condition and reveal who is at risk for it.

Brief to Genetic Mapping

What is genetic mapping?

Developing new and better tools to make gene hunts faster, cheaper and practical for any scientist was a primary goal of the Human Genome Project (HGP).

One of these tools is genetic mapping, the first step in isolating a gene. Genetic mapping - also called linkage mapping - can offer firm evidence that a disease transmitted from parent to child is linked to one or more genes. It also provides clues about which chromosome contains the gene and precisely where it lies on that chromosome.

Genetic maps have been used successfully to find the single gene responsible for relatively rare inherited disorders, like cystic fibrosis and muscular dystrophy. Maps have also become useful in guiding scientists to the many genes that are believed to interact to bring about more common disorders, such as asthma, heart disease, diabetes, cancer and psychiatric conditions.

How do researchers create a genetic map?

To produce a genetic map, researchers collect blood or tissue samples from family members where a certain disease or trait is prevalent. Using various laboratory techniques, the scientists isolate DNA from these samples and examine it for the unique patterns of bases seen only in family members who have the disease or trait. These characteristic molecular patterns are referred to as polymorphisms, or markers.

Before researchers identify the gene responsible for the disease or trait, DNA markers can tell them roughly where the gene is on the chromosome. This is possible because of a genetic process known as recombination. As eggs or sperm develop within a person's body, the 23 pairs of chromosomes within those cells exchange - or recombine - genetic material. If a particular gene is close to a DNA marker, the gene and marker will likely stay together during the recombination process, and be passed on together from parent to child. So, if each family member with a particular disease or trait also inherits a particular DNA marker, chances are high that the gene responsible for the disease lies near that marker.

The more DNA markers there are on a genetic map, the more likely it is that one will be closely linked to a disease gene - and the easier it will be for researchers to zero-in on that gene. One of the first major achievements of the HGP was to develop dense maps of markers spaced evenly across the entire collection of human DNA.

What are genetic markers?

Markers themselves usually consist of DNA that does not contain a gene, however they can tell a researcher the identity of the person a DNA sample came from. This makes markers extremely valuable for tracking inheritance of traits through generations of a family, and markers have also proven useful in criminal investigations and other forensic applications.

Although there are several different types of genetic markers, the type most used on genetic maps today is known as a microsatellite map. However, maps of even higher resolution are being constructed using single-nucleotide polymorphisms, or SNPs (pronounced "snips"). Both types of markers are easy to use with automated laboratory equipment, so researchers can rapidly map a disease or trait in a large number of family members.

The development of high-resolution, easy-to-use genetic maps, coupled with the HGP's successful sequencing and physical mapping of the entire human genome, has revolutionized genetics research. The improved quality of genetic data has reduced the time required to identify a gene from a period of years to, in many cases, a matter of months or even weeks. Genetic mapping data generated by the HGP's laboratories is freely accessible to scientists through databases maintained by the National Institutes of Health and the National Library of Medicine's National Center for Biotechnology Information.

DNA Microchip Technology

What is a DNA microchip?

Scientists know that a mutation - or alteration - in a particular gene's DNA often results in a certain disease. However, it can be very difficult to develop a test to detect these mutations, because most large genes have many regions where mutations can occur. For example, researchers believe that mutations in the genes BRCA1 and BRCA2 cause as many as 60 percent of all cases of hereditary breast and ovarian cancers. But there is not one specific mutation responsible for all of these cases. Researchers have already discovered over 800 different mutations in BRCA1 alone.

The DNA microchip is a mew tool used to identify mutations in genes like BRCA1 and BRCA2. The chip, which consists of a small glass plate encased in plastic, is manufactured somewhat like a computer microchip. On the surface, each chip contains thousands of short, synthetic, single-stranded DNA sequences, which together add up to the normal gene in question.

What is a DNA microchip used for?

Because chip technology is still relatively new, it is currently only a research tool. Scientists use it to conduct large-scale population studies - for example, to determine how often individuals with a particular mutation actually develop breast cancer.

As we gain more insight into the mutations that underlie various diseases, researchers will likely produce new chips to help assess individual risks for developing different cancers as well as heart disease, diabetes and other diseases.

How does a DNA microchip work?

To determine whether an individual possesses a mutation for BRCA1 or BRCA2, a scientist first obtains a sample of DNA from the patient's blood as well as a control sample - one that does not contain a mutation in either gene.

The researcher then denatures the DNA in the samples - a process that separates the two complementary strands of DNA into single-stranded molecules. The next step is to cut the long strands of DNA into smaller, more manageable fragments and then to label each fragment by attaching a fluorescent dye. The individual's DNA is labeled with green dye and the control - or normal - DNA is labeled with red dye. Both sets of labeled DNA are then inserted into the chip and allowed to hybridize - or bind - to the synthetic BRCA1 or BRCA2 DNA on the chip. If the individual does not have a mutation for the gene, both the red and green samples will bind to the sequences on the chip.

If the individual does possess a mutation, the individual's DNA will not bind properly in the region where the mutation is located. The scientist can then examine this area more closely to confirm that a mutation is present.

Last Reviewed: November 3, 2008 （Source：National Human Genomic Program）

DNA Microchips

Perhaps the best-known type of surface attachment involves DNA oligonucleotide microarrays (so called gene chips). Their importance to the study of genetics was illustrated by the appearance in (1999) of an entire supplemental issue of Nature Genetics dedicated to the subject. These chips have been used to enable rapid sequencing of DNA, for the detection of RFLPs (Hacia & Collins, 1999) , for monitoring the presence of microorganisms in the food industry (Kuipers, 1999; Kuipers et al., 1999) , or the environment (Guschin et al., 1997; Oh & Liao, 2000) and to assay gene expression in a wide range of organisms (Harrington et al., 2000) . More recently, chips have been manufactured with a three-dimensional array based on the use of a polyacrylamide matrix (Guschin et al., 1997) . These are likely to lead to faster screening techniques and the use of larger “databases” of genes on the chips for expression studies. Microchip development has also spawned a vast collection of associated industries producing clone-sets, arrayers, scanners and software for data analysis (Epstein & Butlow, 2000) . It seems likely that single chips will get smaller, moving toward the nanoscale and the detection/use of single DNA molecules.

Currently, there are two major variants of DNA chip technology based on the type of immobilised DNA. In the first format, the immobilised probe consists of cDNA (500-5000bp DNA fragments produced by copying expressed genes) spotted onto a surface using robotics. The second format, consists of 20-25-mer oligonucleotides (or peptide nucleic acid - PNA) synthesised in situ (such as the gene chips produced by Affymetrix), or by conventional synthesis and then immobilised. The oligonucleotides are of known DNA sequence allowing detection of complementary sequences. Therefore, the DNA on the chip acts as a probe in a hybridisation reaction (Souteyrand et al., 2000; Southern et al., 1999) . The arrays come in various sizes from macroarrays to microarrays and beyond. Macroarrays are generally produced by hand, or using simple robots to pipette DNA solutions onto prepared surfaces and contains “tens to hundreds” of spots over an area of 500 mm or more. Microarrays contain many thousands of spots over an area of less than 500 mm.

Gene chips for cancer detection

DNA microchips have been used extensively for research purposes, but they also have a powerful use as a biosensor. For example, the onset of prostate cancer involves the differential expression of approximately 500 genes. A prototype chip for prostate cancer detection is already under development by the biotechnology firm Darwin Molecular (Bothell, Washington USA). They predict that within a year they will have a system that can distinguish slow-growing cancers that do not require immediate treatment and those aggressive tumours that require surgery (Zajtchuk, 1999).

Detection of hybridisation usually involves the use of PCR amplified, fluorescently-tagged DNA. Generally, for gene expression studies, this involves amplification of a pair of mRNA samples (i.e. a control and an experimental sample) using different fluorophores for each sample. Identical amounts of the amplified cDNA are hybridised to the same DNA chip and fluorescent intensities compared (Winzeler et al., 1999) . The use of two fluorophores overcomes problems associated with different amounts of probe DNA deposited at the specific spots on the chip.

The first consideration in the design of a DNA chip is the support material and the production of simple arrays in a research environment was discussed by Cheung et al. (1999) . Most usually, this involves the use of glass. However, recently silicon has also been employed as a support allowing the use of its semiconducting properties (Souteyrand et al., 2000) . Electrochemical deposition of oligonucleotides, via pyrrole groups is used to attach the DNA to the surface of electrodes (Livache et al., 1998a) . These electrodes can interact with the silicon allowing for the development of new techniques for detecting hybridisation based on the semiconductor properties of the silicon. A typical 128-sample grid is shown in Figure 17. The chip uses polarisation of the electrodes (Souteyrand et al., 2000) to prevent diffusion, allowing the samples to concentrate ensuring hybridisation occurs in a few seconds. After hybridisation, a short burst of the opposite polarisation will release weakly bound molecules improving signal-noise ratios. Finally, the charge on the DNA strands allows detection of hybridisation without using fluorophores, through the field-effects on the electrodes.

Perhaps the company most associated with DNA microchips, Affymetrix, believes that the capacity of DNA microchips is following Moore’s Law, which predicts a two-fold increase in computer chip memory every 18 months. Affymetrix’s prototype chips held 20,000 oligonucleotide DNA probes. By 1997, this number had increased to 65,000 probes and it has recently introduced a chip with 400,000 probes. Ten such chips could scan the entire human genome for ‘malfunctioning’ genes.

（Source：Keith Firman, Nanonet)