How to Make Array CGH

How to Make Array CGH

Method

Reference: Wicker, etc (2007)

The first must come out the embryo chromosome DNA extract, and in the sign the green fluorescence treats as the experimental group, the comparison group collects a normal person's corpuscular cell, in half cell extract DNA and sign red fluorescence, another one half in microslides on can catch the blue color the fluorescence. The experimental group and the comparison group competes template, when the experimental group and the comparison group simultaneously join Cot-1 DNA to be able to reduce non- specificity DNA the union, uses the computer under the fluorescence microscope image to analyze again, may aim at the entire 23 pair of chromosomes does comprehensively compared to, that is may simultaneously detect 23 pair of chromosomes unusual, only cannot aim at some specific DNA fragment like FISH to make the analysis. At present further develops the competition gene micro array analytic method (array-CGH) Using the gene chip did comprehensively to the entire 23 pair of chromosomes compared to, its analysis reaches as high as 100 to arrive 200K bp, compared with traditional CGH analysis higher, but the cost is soaring, still has not reached the clinical practical boundary.

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Introduction to Array CGH

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Reference theses

Reference: Barrett MT. (2004)

For CGH hybridization, need following procedures:

1. Reference sample (46,XX female) 20 µg of genomic DNA.

2. Experimental sample with AluI (20 units) and RsaI (20 units) (Promega).

3. Amplified: 10 ng of genomic DNA from the reference and experimental sample.

4. All done for a minimum of 2 h at 37°C and then verified by agarose gel analysis.

Individual reference and experimental samples were then filtered by using the QIAQuick

PCR clean-up kit (Qiagen).

5. Labeling reactions were performed with 6 µg of purified restricted DNA and a Bioprime

labeling kit (Invitrogen) according to the manufacturer’s instructions in a volume of 50 µl

with a modified dNTP pool containing 120 µM each of dATP, dGTP, and dCTP; 60

µM dTTP; and 60µM Cy5-dUTP (for the experimental sample) or Cy3-dUTP (for the

46,XX female reference) (PerkinElmer).

6. Experimental and reference targets for each hybridization were pooled and mixed with

50µg of human Cot-1 DNA (Invitrogen) /100 µg of yeast tRNA (Invitrogen)_1_

hybridization control targets (SP310, Operon Technologies, Alameda, CA).

7. The target mixture was purified, concentrated the final volume of 250 µl, and then mixed

with an equal volume of Agilent 2µ in situ hybridization buffer.

8. Hybridization to the array (before that need some complex procedure).

Human Cot-1 DNA is gained from human placental DNA by extracting, shearing, denaturing, and reannealing DNA under conditions that enhence for repetitive DNA sequences such as the Alu I and Kpn I families .Human Cot-1 DNA can reduce cross-hybridization to human repetitive DNA when human DNA probes (i.e., cosmids, YACs, and chromosome-painting probes) are hybridized in situ during filter hybridization experiments. Human Cot-1 DNA which can be labeled to provide an effective hybridization probe to check for the presence of human DNA.

Using the DNA probe by labeled the fluorescence of the hybridization process, to make the location of gene or DNA on the chromosome.