Extracting DNA from Whole Blood

Extracting DNA from Whole BloodGroup 4 - 4Bio2Binag, Sam DominicCaringal, Joe Mari IsabellaChiang, RowenaVerin, MaryneilIntroductionDNA EXTRACTIONprocess of purification of DNA from sample using a combination of chemical and physical methods one of the most widely used molecular biology techniques usually the first step in a longer laboratory process IntroductionDNA EXTRACTIONimportant routine procedure in the fields of biotechnology and forensics allow scientists to detect genetic diseases, analyze forensic evidence, produce DNA fingerprint of individuals, and create of genetically engineered organisms that can produce beneficial products. IntroductionDNA EXTRACTION OVERVIEWlyse or break open the cell to expose the DNA of interest.

washing with detergent

precipitation and resuspending of DNA

IntroductionBLOODexcellent source of human DNA

Friedrich Miescher- isolated the first crude extract of DNA from white blood cells in 1869- discovered the substance nuclein now known as DNA

IntroductionBloodWhole blood contains three types of cells: red blood cells (RBCs), white blood cells (WBCs), and platelets. RBCs do not have any DNA

WBCs contain DNAObjectivesTo prepare a flow diagram of the experimental protocolTo extract DNA from blood

HypothesisDifferent DNA sources produce different amounts of DNA and the amount and freshness of the blood affect the quality and quantity of the DNA obtained.

MethodologyResults and DiscussionCell ConcentrationCentrifugationPhosphate Buffered Saline (PBS)

Cell LysisSTE, SDS, Proteinase KResults and DiscussionSTESodium chloride: for DNA to precipitateTris: buffer systemEDTA: chelates cationsSDSDetergentDisruption of cell membranesEmulsification of lipids and proteinsResults and DiscussionProteinase KDenatures proteinsDisables nucleases

DNA ExtractionPhenol-chloroform extractionLiquid-liquid extractionFor isolation of DNA, RNA, proteinsResults and DiscussionForms lower organic phase and upper aqueous phase

Results and DiscussionAqueous PhaseContains hydrophilic DNA

Organic PhaseContains hydrophobic lipids

White interphaseInteractions of hydrophobic molecules of proteins with phenolResults and DiscussionPhenol: constitutes lower organic phaseChloroformMiscible with phenolForces a sharper separation between organic and aqueous phaseIsoamyl alcoholAdded in small amountsPrevents foamingResults and DiscussionDNA Concentration (Washing)Sodium acetate (Salt)Makes DNA less soluble in the aqueous solutionMore efficient precipitation of DNA in alcohol

Ethanol Precipitation

Washing with 70% EthanolWash away residual saltsResults and DiscussionTE BufferTris-EDTA BufferSolubilize DNA while preventing degradationGuide Questions1. What is the role of the following in DNA extraction?a. Ethanolb. NaClc. SDSd. TE Buffere. EDTAGuide QuestionsEthanolEthanol is used in DNA extraction to force the DNA to precipitate in a solution. Ethanol prevents the DNA from dissolving into the water, instead causing it to precipitate out so it can be separated and extracted. Without the addition of ethanol, DNA will just dissolve in water.

Guide Questionsb. NaClSalt neutralizes the charges on the sugar phosphate backbone. The positively charged sodium ions neutralize the negative charge on the PO3- groups on the nucleic acids, making the molecule far less hydrophilic, and therefore much less soluble in water.

Guide Questionsc. SDS

SDS is a strong anionic detergent that can solubilize the proteins and lipids that form the membranes. This will help the cell membranes and nuclear envelopes to break down and expose the chromosomes that contain the DNA. In addition to removing the membrane barriers, SDS helps release the DNA from histones and other DNA binding proteins by denaturing them.

Guide Questionsd. TE Buffer

The purpose of TE buffer is to solubilize DNA or RNA, while protecting it from degradation.

Guide Questionse. EDTA

EDTA behaves as chelating agent for divalent ions like Ca++ and Mg++. Ca++ ions are initiator of blood clotting and behave as cofactor for clotting enzymes. Hence, EDTA prevents blood coagulation by chelating these ions.

Guide Questions2.Give the complete chemical names of the following:a. SDS Sodium dodecyl sulfateb.Tris Buffer- Tris(Hydroxymethyl)aminomethane

c. EDTA - Ethylenediaminetetraacetic acid

Guide Questions3. Why should the extracted DNA be immersed in TE buffer? TE buffer protects DNA from degradation. "TE" stands for its components Tris and EDTA. Tris serves as a buffering agent while EDTA serves as a chelating agent. TE buffer is commonly used to protect the DNA while storing it.

ConclusionThe purpose of DNA extraction is to obtain DNA in a relatively purified form, which can be used for further investigations. An efficient DNA extraction method for whole blood was carried out using phenol-chloroform extraction process.The protocol designed for blood yielded DNA. The number, age, and freshness of the source affect the quality and quantity of the DNA obtained.

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