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.

Y-Chromosome Analysis

Since the Y chromosome passes from a male to his son, analysing genetic markers on a Y chromosome can be of aid in identifying familial ties in males or for analysing any evidence entailing many males. Another benefit of Y-chromosome analysis is to establish a family line over many generations.

There are other types of analysis but these are some of the main traditional and current methods used to analyse DNA. No doubt, new techniques will be developed that will be even more rapid, successful and cost-effective.

Mitochondrial DNA Analysis

Mitochondrial DNA analysis works well on samples that are unable to be analysed through RFLP or STR analysis. There are two kinds of DNA in the cell - mitochondrial DNA and nuclear DNA. With other types of analysis, nuclear DNA is removed from the sample but with mitochondrial DNA analysis, DNA is removed from the cell's mitochondria. Sometimes, a sample can be old and will no longer have nuclear material in the cell, which poses a problem for the other types of DNA analysis. With mitochondrial DNA analysis, however, mitochondrial DNA can be removed, thus having important ramifications for cases that were not solved over many years. This means that mitochondrial DNA analysis can be very valuable in investigations for a missing person. Mitochondrial DNA will be the same from a woman to her daughter because it is passed on from the egg cell.

Recombinant DNA and genetic techniques

Recombinant DNA (or rDNA) is made by combining DNA from two or more sources. In practice, the process often involves combining the DNA of different organisms. The process depends on the ability to cut and re-join DNA molecules at points which are identified by specific sequences of nucleotide bases called restriction sites. DNA fragments are cut out of their normal position in the chromosome using restriction enzymes (also called restriction endonucleases) and then inserted into other chromosomes or DNA molecules using enzymes called ligases.

Gene Cloning

This describes the process of copying fragments of DNA which can then be used for many different purposes, such as creating GM crops, or finding a cure for disease.  There are two types of gene cloning:  in vivo, which involves the use of restriction enzymes and ligases using vectors and cloning the fragments into host cells (as can be seen in the image above).  The other type is in vitro which is using the polymerase chain reaction (PCR) method to create copies of fragments of DNA.

For in vivo cloning a fragment of DNA, containing a single gene or a number of genes, is inserted into a vector that can be amplified within another host cell. A vector is a section of DNA that can incorporate another DNA fragment without losing the capacity for self-replication, and a vector containing an additional DNA fragment is known as a hybrid vector. If the fragment of DNA includes one or more genes the process is referred to as gene cloning.

Cloning DNA in Plasmids

Contributor: Genome Management Information System, Oak Ridge National Laboratory, U.S. Department of Energy Genome Programs  http://genomics.energy.gov

There are 4 different type of vectors:

• Plasmid vectors
• Lamda (λ) phage vectors
• Cosmids
• Expression vectors

The host cell copies the cloned DNA using its own replication mechanisms. A variety of cell types are used as hosts, including bacteria, yeast cells and mammalian cells.

Polymerase Chain Reaction (PCR)

Source:  Andy Vierstraete 

http://users.ugent.be/~avierstr/principles/pcr.html

This is an in vitro method for making many copies of a specific section of DNA, without the need for vectors or host cells.  The DNA to be copied – the template DNA – is mixed with forward and reverse primers complementary to the end of the template DNA, nucleotides, and a version of DNA polymerase known as Taq polymerase. (This enzyme is stable under high temperatures, and is obtained from the thermophilic bacterium Thermus aquaticus.) The process involves the repetition of three steps:

• denaturation, which separates the two nucleotide strands of the DNA molecule
• primer annealing, in which the primers bind to the single-stranded DNA
• extension, in which nucleotides are added to the primers – in the 5' to 3' direction – to form a double-stranded copy of the target DNA.

Each cycle takes a few minutes, and repeated cycles can produce large amounts of a specific DNA sequence in a matter of hours rather than days. However, this cloning method does require knowledge of some details about the nucleotide sequence to be copied, and the technique is very sensitive to small amounts of contamination.

Gene Libraries

Source: http://www.emunix.emich.edu/~rwinning/genetics/index.htm

A gene library is a large collection of cloned DNA sequences from a single genome.  A genomic library, (as can be seen above) in theory, would contain at least one copy of every sequence in an organism’s genome. These are used to investigate the structure of a given chromosome, or to clone specific genes.  These types of libraries may be prepared from a subset of the entire genome (for example, a single chromosome). The first step in creating a genomic library is to break up, or ‘fractionate’, the genome using physical methods or restriction enzymes. The fragments are then linked to appropriate vectors and cloned in a suitable host cell population.

A cDNA library (complementary DNA) contains DNA present in a given cell population which is prepared from the mRNA (messenger RNA) using the enzyme reverse transcriptase.  The resulting cDNA represents the genes expressed in the cell population as a subset of the entire genome, and can be cloned using a vector and suitable host cell (as seen in the diagram above). The cDNA will not include introns or regulatory sequences as these are removed from the RNA during processing, and this makes a cDNA library easier to maintain.  A cDNA library can also be prepared using reverse transcriptase PCR (RT-PCR).

The Identification of Gene Products in a Gene Library

Restriction enzymes (to cut the DNA) and gel electrophoresis (to separate the resulting fragments) can be used to produce a physical map of DNA segments in a process known asrestriction mapping.  An example of what one of these may look like can be seen below.

Source: University of Leicester

There are also a number of techniques that can be used to identify specific genes or gene products within a gene library and these are: Southern blotting, Northern blotting andWestern blotting. However, the most powerful experimental technique for investigating genetics at the molecular level is DNA sequencing, which allows the nucleotide sequences of genes – even whole chromosomes – to be determined. Automated sequencing technologies are now allowing us to sequence the entire genomes of organisms from bacteria to human beings.

Molecular Genetics and Biotechnology

The new techniques of molecular genetics, combined with developments in associated biotechnologies, have led to advances in a number of different fields. We can now analyse the genomes of species that make an important contribution to agriculture, fuel production or drug development. We can move specific genes from one organism to another to createtransgenic plants and animals, and use animal cloning techniques to produce animals that are genetically identical, such as Dolly the sheep, and more recently, cloned pets such as cats and dogs.

The process of cloning is straightforward, but the results are not always predictable.  It took many hundreds of attempts to get it to work and produce one live sheep, and cloning in itself raises many questions not only about benefits and risks but also many ethical questions.

Diagram of pigs to show how animal cloning is carried out.  Source: National Human Genome Research Institute

The technique of genetic fingerprinting, which enables the identification of individuals and the relationships between individuals has found many applications in science today. There is also ongoing research into gene therapy which examines the possibility of introducing cloned genes to compensate for defective, mutant genes. And other areas, for example,human cloning and stem cell research open up many ethical issues that must be addressed alongside the scientific developments.