DNA sequencing and genomics

One of the most fundamental technologies developed to study genetics, DNA sequencing allows researchers to determine the sequence of nucleotides in DNA fragments Developed in 1977 by Frederick Sanger and coworkers, the sequence of chain termination is now routinely used for DNA fragments With this technology, researchers were able to study the molecular sequences associated with many human diseases

As sequencing has become cheaper, the researchers have been sequencing genomes of many organisms, using computational tools to put together a number of different fragments (a process called genome assembly) This technology is used to sequence the human genome, leading to the completion of the Human Genome Project in 2003 The new high-throughput sequencing technology dramatically lowering the cost of DNA sequencing, with many researchers hope to bring the human genome resequencing cost a thousand dollars

The large amount of sequence data available has created the field of genomics, research that uses computational tools to search and analyze patterns in the genome of an organism is full Genomics can also be regarded as a subfield of bioinformatics, which uses computational approaches to analyze large sets of biological data

Extraction of DNA from Fresh Bone

Take a sample of nucleic acids like DNA from the bones to analyze gene expression in the search for somatic mutations in diseased tissues or other tumors, or to genotype archive material, when other sources of DNA are not available You can use a different kit that has been provided by biotechnology companies But if you want to extract a DNA sample from a large amount, you can use artificial methods, as described here to be cost effective


There are four procedures to ensure that the successful extraction of nucleic acids from the network:
1 disrupt the fabric so that the reagent for extraction can reach the cells

2 interference with the cell membrane so that the nucleic acids are released

3 nucleic acid separation from other cellular components

4 nucleic acid precipitation and volatilization

Material
1 DNA extraction buffer: Add 176 mol of 075 M sodium citrate, pH 70, 264 mol 10% sodium laurel seriously, and 250 g of guanidine isothiocyanate in 293 ml of distilled water and stir well Add 72 micro liters lyses buffer beta-mercaptoethanol/mL day usage
All chemicals must be capable of molecular biology Solutions can be stored at 4oC for 3 months

2 05 M ETDA: Add 9305 g EDTA to 300 ml of distilled water and add 10 N Noah, pH 80 Bring up to 500 ml Autoclave

Tries-EDTA: Add 1 ml of 1 M Tries to 200 micro liters 05 M EDTA To 100 ml with distilled water

3 3 M sodium acetate, pH 52: Add 4018 g sodium acetate per 800 ml of distilled water Adjust pH to 51 with glacial acetic acid Bring to 1 L with distilled water Autoclave

4 General reagents: Tries-saturated phenol pH 78 to 80 (Sigma), chloroform, ethanol 100% isopropanol

Method
1 Collect samples of bone in a sterile container containing buffered saline (PBS) and transport to the laboratory within 1-2 h
If DNA extraction is not started immediately, freeze the samples at-20oC or below for later use

2 Place bones in a clean glass dish plate With bone cutters or sharp scissors strong, isolated piece of bone about 1 cm3, and transfer it to bijoux 5 ml clean

3 Add 1 mol DNA extraction buffer and homogenize the tissue with scissors until a solution is obtained in mud

4 Transfer 500 micro liters spare screw cap conical mud in 15 mol Expender tubes

5 Add a volume of Tries-saturated phenol, followed by the volume of chloroform per tube Mix by inverting the tube several times or agitation Do not vortex, as long grooves cause the vortex-mixing of DNA cutting

6 Centrifuge tubes at 10 000 g for 20 minutes to separate phases

7 Transfer to the upper layer of fresh centrifuge tube (with respect to volume), taking care not to disturb the layer of milk on the interface Repeat steps 5-7 if the interface is disturbed

8 Add a volume of cold isopropanol and 01 volume of 3 M sodium acetate to the supernatant Stir well and let stand for 15 minutes on the ice

9 Centrifuge tubes at 10 000 g for 20 minutes to pellet DNA
East Expender tube to identify where is the DNA pellet Pellets will be visible at the bottom of the tube

10 Aspirate and discard supernatant, being careful not to disturb the sediment Wash the sample with 175 ml ice-cold ethanol and centrifuged at 10 000 g for 5 minutes Aspirate and discard supernatant and repeat wash

11 Dissolve the DNA pellet 10-50 micro liters of water or Tries-EDTA (you can pool the DNA from the sample at this stage) and measured by spectrophotometer or with Hoechst 33 258
Hoechst 33 258 is a DNA-specific dye that can be used to measure DNA

12 Store samples frozen at-20oC or lower