Gene therapy

Gene therapy involves supplying a functional gene to cells lacking that function, with the aim of correcting a genetic disorder or acquired disease. Gene therapy can be broadly divided into two categories. The first is alteration of germ cells, that is, sperm or eggs, which results in a permanent genetic change for the whole organism and subsequent generations. This “germ line gene therapy” is considered by many to be unethical in human beings. The second type of gene therapy, “somatic cell gene therapy”, is analogous to an organ transplant. In this case, one or more specific tissues are targeted by direct treatment or by removal of the tissue, addition of the therapeutic gene or genes in the laboratory, and return of the treated cells to the patient. Clinical trials of somatic cell gene therapy began in the late 1990s, mostly for the treatment of cancers and blood, liver, and lung disorders.

Despite a great deal of publicity and promises, the history of human gene therapy has been characterized by relatively limited success. The effect of introducing a gene into cells often promotes only partial and/or transient relief from the symptoms of the disease being treated. Some gene therapy trial patients have suffered adverse consequences of the treatment itself, including deaths. In some cases, the adverse effects result from disruption of essential genes within the patient's genome by insertional inactivation. In others, viral vectors used for gene therapy have been contaminated with infectious virus. Nevertheless, gene therapy is still held to be a promising future area of medicine, and is an area where there is a significant level of research and development activity.

Epigenetics Protocols

Background of Epigenetics:

There is far more to genetics than the sequence of building blocks in the DNA molecules that make up our genes and chromosomes. The "more" is known as epigenetics.

What is epigenetics?
Epigenetics, literally "on" genes, refers to all modifications to genes other than changes in the DNA sequence itself. Epigenetic modifications include addition of molecules, like methyl groups, to the DNA backbone. Adding these groups changes the appearance and structure of DNA, altering how a gene can interact with important interpreting (transcribing) molecules in the cell's nucleus.

How do epigenetic modifications affect genes?
Genes carry the blueprints to make proteins in the cell. The DNA sequence of a gene is transcribed into RNA, which is then translated into the sequence of a protein. Every cell in the body has the same genetic information; what makes cells, tissues and organs different is that different sets of genes are turned on or expressed.

Epigenome NoE - Protocols Database

Epigenetics Protocols Database. The following protocols are selected, solicited and ... Protocol Online �� Epigenetic Station's Top Hits �� Cold Spring Harbor ...
www.epigenome-noe.net/researchtools/protocols.php 

Protocols- Epigenetics and Methylation Protocols > Bisulfite ...

Bisulfite Treatment Methylation Assay Protocol. Bisulfite Methylation. ... or epigenetic information differs on the two complementary strands of DNA. ...
www.molecularstation.com/.../Bisulfite_Treatment_Methylation_Assay_Protocol/

DNA Methylation in Cancer - Protocols - Southern Blot Analysis ...

Care Centers & Departments, --, Care Center Information, A to Z Department List, --, Brain & SpineCenter, Breast Center ...
www.mdanderson.org/departments/methylation/display.cfm?id=816A773B-F17B-43D4-9B0090D858EED92B&method=...

DNA methylation analyis using restriction enzyme digestion

This site contains protocols, product information and other material for molecular biologists, biochemists, immunologists, and all people interested in ...
www.methods.info/Methods/DNA_methylation/Restriction_analysis.html -

EpiTYPER for Quantitative DNA Methylation Analysis | Protocol ...

Protocol Archive. BioTechniques Online ... Epigenetics. Epigenetic Modification Analysis ... Return to table of contents for the Protocol Guide ...
www.biotechniques.com/default.asp?page=protocol&subsection=article_display&id=112658

Epigenome NoE - protocol: DNA Methylation Analysis by Bisulfite ...

Genomic DNA methylation is one of the most important epigenetic modifications in eukaryotes. It is essential for life and its alteration is often associated ...
www.epigenetics-noe.net/researchtools/protocol.php?protid=34

CSH Protocols -- Collected Res9

DNA Modification/Epigenetics. Protocols 1-10 of 13 total displayed. ... Protocol Icon. DNA Immunoprecipitation (DIP) for the Determination of DNA-Binding ...
www.cshprotocols.org/cgi/collection/dna_modification_epigenetics

Epigenetics- Top Hits

Chromosome Conformation Capture (3C) Protocol developed by Alice Horton .... The Epigenetics Journal publishes original research and reviews covering ...
epigeneticstation.com/epigenetic-links/index.php?list=top  

Nature Protocols: DNA methylation analysis by pyrosequencing

This Protocol is listed in the following Categories: Gene expression. Author(s): J?rg Tost Affiliation(s): Laboratory for Epigenetics, CEA-Institut de ...
www.natureprotocols.com/2007/09/06/dna_methylation_analysis_by_py.php

Immunolabelling, immunolabeling and immunostaining of proteins in ...

Science. Retroelements �� Pararetrovirus �� Bio banana Musa genomics �� Microarrays �� Repetitive DNA �� Chromosome Model �� Bovid (cow) diversity and evolution ...
www.le.ac.uk/bl/phh4/immunopr.htm  

DNA Methylation in Cancer - Protocols - DNA-Methyltransferase ...

Care Centers & Departments, --, Care Center Information, A to Z Department List, -, Brain & SpineCenter, Breast Center ...
www.mdanderson.org/departments/methylation/display.cfm?id=9B5B6082-C53A-4A86-BEE3EE149A52251E&method=...

Matthew J. Oberley, Peggy J. Farnham McArdle Laboratory for Cancer ...
Matthew J. Oberley, Peggy J. Farnham. 1. McArdle Laboratory for Cancer Research. University of Wisconsin Medical School. 1400 University Ave ...
www.genomecenter.ucdavis.edu/farnham/protocols/MOJO%20Dec%202003%20ChIP-chip%20protocol.pdf

WWW Virtual Library: Drosophila Protocols

A listing of protocols in Drosophila (fruit fly) research available on the Web.
www.ceolas.org/VL/fly/protocols.html  

DNA Methylation Analysis Using Restriction Enzyme Digestion

This site contains protocols, product information and other material for molecular biologists, biochemists, immunologists, and all people interested in ...
www.methods.info/Methods/DNA_methylation/Restriction_analysis.html -

DNA methylation analysis using bisulphite sequencing

This site contains protocols, product information and other material for molecular biologists, biochemists, immunologists, and all people interested in ...
www.methods.info/Methods/DNA_methylation/Bisulphite_sequencing.html -

DNA methylation analysis using Single Nucleotide Primer Extension (SNuPE)

This site contains protocols, product information and other material for molecular biologists, biochemists, immunologists, and all people interested in ...
www.methods.info/Methods/DNA_methylation/SNuPE.html -

Cancer Genetics in the 21st Century - National Cancer Institute (Video)

Between these Dates:. Jan. Feb. Mar. Apr. May, Jun. Jul. Aug. Sept. Oct. Nov. Dec. 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008 ...
www.nci.nih.gov/newscenter/benchmarks-vol5-issue1/Video

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The Google theme is full of errors and when I report these errors Rojin P Mani (who seems to be the only person running the site) said "Sorry I can not get to the office today".

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DNA MICROARRAY PROTOCOL

i)           Set-up the following Pre-Hybridisation solution in a Coplin Jar and        incubate at 65°C during the labeling incubation period to equilibrate. 20X SSC 8.75 ml 20% SDS 0.25 ml BSA (100 mg/ml) 5.0 ml H2O to 50.0 ml

ii)            Label control and test genomic DNA as follows:- CONTROL TEST Genomic DNA ˜ 2 mg ˜ 2 mg Random Hexamers (3 mg/ml) 1 ml 1 ml H2O to 41.5 ml to 41.5 ml Heat at 95ºC for 5 minutes. Snap cool on ice and briefly centrifuge. 10X buffer 5 ml 5 ml dNTP's (5mM each dATP, dGTP & dTTP, 2mM dCTP) 1 ml 1 ml Cy-labelled dCTP 1.5 ml (Cy3) 1.5 ml (Cy5) Klenow fragment (10U/ml) 1 ml 1 ml Incubate at 37°C for 90 minutes.

iii)          Incubate the microarray slide(s) in the Pre-Hybridisation solution for 20 minutes at 65°C, beginning just before the end of the labelling reactions incubation time at 37°C.

iv)          Combine the control and test reactions and purify using the Qiagen MinElute PCR Purification kit, using a two step wash stage using 500 ml then 250 ml volumes of Buffer PE and eluting the labeled cDNA from the MinElute column with 14 ml H2O. The columns retain approximately 1 ml, so the final eluted volume will be 13 ml.

v)           Rinse the pre-hybridised microarray slides in H2O for 1 minute, then in isopropanol for 1 minute. Spin at 1500 rpm for 5 minutes to dry slides. Keep in covered slide box. 1 NICK DORRELL - LAST UPDATE FEBRUARY 2004

vi)          Prepare the Hybridisation solution as follows: - Sample 13 ml H2O 26 ml 20X SSC 12 ml 2% SDS 9 ml Heat at 95ºC for 2 minutes. Allow to cool slowly at room temperature and centrifuge for 30 seconds. Add 2 x 20 ml H2O to the corners of the hybridisation chamber. Place a slide into the chamber. Place a LifterSlip™ glass coverslip (22 mm x 25 mm) over the array section on the slide using tweezers. Pipette the Hybridisation solution onto the slide at the top of the coverslip. Seal the chamber and incubate in a water bath at 65°C overnight.

vii)         Prepare Wash solutions as follows: - Wash A (1X SSC 0.5% SDS) Wash B (0.06X SSC) 20X SSC 20 ml 2.4 ml 20% SDS 1 ml H2O to 400 ml to 800 ml Incubate Wash A solution at 65ºC overnight. Dispense 400 ml volumes into three glass slide washing dishes. Remove slide(s) from the hybridisation chambers and gently remove coverslip(s) by rinsing in Wash A. Place slide(s) in a slide rack and rinse with agitation for 5 minutes. Transfer slide(s) to a clean slide rack and rinse with agitation in Wash B(i) for 2 minutes, then in Wash B (ii) for a further 2 minutes. Spin at 1500 rpm for 5 minutes to dry slide(s).

viii)      Scan slide(s) using Affymetrix 418 scanner and analyse data

（NICK DORRELL - LAST UPDATE FEBRUARY 2004）

Polymerase Chain Reaction (PCR) Analysis

PCR analysis is a technique that allows technicians to create millions of precise DNA replications from a single sample of DNA. In fact, DNA amplification alongside PCR can let forensic scientists perform DNA analysis on samples that are as tiny as only a couple of skin cells. In contrast to some other DNA analysis techniques, PCR analysis has the advantage of analysing minuscule sample sizes, even if they are degraded although they must not be contaminated with DNA from other sources during the collection, storage and transport of the sample.

Restriction Fragment Length Polymorphism (RFLP)

RFLP is a technique that is not widely used now but it was one of the first techniques used for DNA analysis in forensic science. Large sample sizes are needed for RFLP relative to newer techniques - usually a sample would need to be approximately the size of a one-pound coin. While that in itself may sound small, it is large relative to other techniques such as PCR analysis that require only a few cells for successful sequencing. In RFLP, the different lengths of DNA fragments are analysed. These fragments are from the digestion of a sample of DNA with a restriction endonuclease enzyme. The enzyme chops DNA in a certain style - the restriction endonuclease recognition site. Whether or not particular recognition sites are present will provide different lengths of DNA fragments, which are then divided up through electrophoresis. DNA probes then serve to hybridise the fragments through complementary binding.

Short Tandem Repeat (STR) Analysis

STR analysis works to examine individual areas in DNA. The differences from the collective areas of one person to another can allow for distinguishing between individuals. In criminal investigations, there are thirteen regions that are analysed and compared to establish profiles. In fact, DNA databases used at the government level involve the sequence of these thirteen regions. The chances of two people having the exact same thirteen regions is virtually impossible - likely one in a billion. A common DNA joke is that a person's odds of winning the lottery are higher than finding a perfect match for the thirteen regions.