Nucleic Acid Isolation Lecture Notes PDF

Nucleic acid isolation from clinical specimens PRESENTED BY Thitima Sumphanapai, PhD Division of Clinical Hematology and Microscopy Department of Medical Technology, School of Allied Health Sciences, University of Phayao ...

Nucleic acid isolation from clinical specimens PRESENTED BY Thitima Sumphanapai, PhD Division of Clinical Hematology and Microscopy Department of Medical Technology, School of Allied Health Sciences, University of Phayao  Learning outcomes Be able to explain the methods for nucleic acid extraction Be able to explain the steps for nucleic acid extraction Be able to explain the properties of chemical used in nucleic acid extraction  Types of specimen Blood Cells Microorganisms Whole blood Swab จากกระพุ้งแก้ม Virus Buffy coat เซลล์จากสารคัดหลั่ง Bacteria Fungus  Genomic DNA extraction methods Precipitation/ Suspension Cell lysis Extraction Washing /Elution Lysis buffer Phenol Chroloform Isopropanol/Ethanol TE buffer Salting-Out Method Solid-Phase Extraction  Lysis buffer Components of lysis buffer in genomic DNA extraction Denature proteins, release DNA, minimizing contamination from RNA and other cellular components If DNA extraction is performed on whole blood>>>>hemolysis step by hypotonic buffer to reduce the amount of hemoglobin in a test sample Guanidine thiocyanate, which helps in the denaturation of proteins and the disruption of cell membranes. Detergents such as Triton X-100 or SDS, which aid in the solubilization of cell membranes and the release of DNA Protease K, an enzyme that helps in the digestion of proteins and the removal of contaminants. RNase A or RNase H to degrade RNA and prevent its contamination in the final DNA preparation.  Phenol Chloroform Extraction Phenol and chloroform are organic solvents that separate DNA or RNA from contaminants based on their different solubilities. A gold-standard method to produce high-purity DNA Organic chemistry extraction Phenol/Chloroform/Isoamyl alcohol (25:24:1) Phenol: strong affinity for proteins, denature them and precipitate out of the solution. Chloroform: used to separate the aqueous phase from the organic phase. Isoamyl alcohol: reduce foaming and ensure deactivation of RNases.  Phenol Chloroform Extraction DNA Precipitation: add ethanol or isopropanol to the solution. This causes the DNA to precipitate out of the solution due to its lower solubility in alcohol. Washing: The DNA pellet is washed with ethanol to remove any remaining contaminants and salts. Finally, the DNA is resuspended in an appropriate buffer or water, making it ready for downstream applications.  Phenol Chloroform Extraction Labor-intensive and requires some user expertise. Time consuming Involves multiple centrifugation steps Transferring solutions from one container to another so if process in many sample it is risk of specimen mix-ups. Phenol-chloroform solutions are chemical hazards. Residual phenol and/or chloroform contaminant will inhibit downstream enzymatic reactions  Salting-Out Method As an alternative to the phenol-chloroform extraction procedure The salting-out method has the advantage of not using toxic chemicals. At high salt, proteins are dehydrated, lose solubility, and precipitation The use of salt: Sodium Chloride, Potassium acetate, Ammonium acetate The major limitation is the purity  Protein Precipitation Centrifugation Cell lysis Protein digestion (Salt) (remove protein) DNA resuspension DNA precipitation (TE buffer) (Ethanol) DNA in supernatant  Solid-Phase Extraction DNA selectively binds to a solid-phase matrix (e.g., silica, magnetic beads) while contaminants are removed. Negatively charged DNA binds to a positively charged silica membrane and hold it during centrifugation  Solid-Phase Extraction Nowadays, all the DNA extraction kits available based on this method Unlike previous methods, the overall yield and concentration of DNA prepared are much lower. This method prepares 10ug or less DNA with concentrations in the range of 20 to 80ng/uL Sample requires in this method uses 200 to 500 ul Binding buffer Washing buffer Elution buffer  Solid-Phase Extraction Binding buffer Chaotropic salts (guanidine isothiocyanate or ammonium acetate) These salts help to disrupt hydrogen bonds and hydrophobic interactions between DNA molecules, allowing DNA to bind more effectively to the solid-phase matrix. pH Adjusting Agents (Tris-HCl) Maintain the optimal pH for DNA binding Washing buffer is designed to remove impurities, proteins, and salts while retaining the purified DNA on the solid-phase matrix Alcohol (Ethanol or Isopropanol) help precipitate proteins and other contaminants promotes the separation of proteins from DNA  Solid-Phase Extraction Elution buffer is designed to disrupt the binding of DNA to the solid-phase matrix and release the purified DNA for collection. Low-Salt Buffer (Tris-HCl) helps to reduce the electrostatic interactions between DNA and the solid-phase matrix, allowing DNA to be released. Nuclease-Free Water It is essential to use nuclease-free water to avoid DNA degradation by nucleases. EDTA prevent DNA degradation by DNases  Overview of common DNA extraction protocols Schmitz, TC, Eren, AD, Spierings, J, de Boer, J, Ito, K, Foolen, J. Solid-phase silica-based extraction leads to underestimation of residual DNA in decellularized tissues. Xenotransplantation. 2021; 28:e12643 Phenol-Chloroform Method Salting-Out Method Solid-Phase Extraction Advantages: Advantages: Advantages: High purity Simplicity High purity Suitable for large-scale Cost-effective Automation-friendly DNA isolation Suitable for small-scale Consistent results Disadvantages: DNA extraction Disadvantages: Hazardous chemicals Disadvantages: Cost Labor-intensive Lower purity Sample size limitations Time-consuming Limited to specific Choice of matrix Limited automation sample types affects performance Less effective for complex or impure samples  RNA extraction methods Precipitation Cell lysis Extraction Suspension/Elution /Washing Lysis buffer Chloroform TE buffer, Isopropanol/Ethanol DEPC-treated water Solid-Phase Extraction  Lysis buffer Components of lysis buffer in RNA extraction Trizol: Guanidine thiocyanate + Phenol, disrupts the cellular and molecular structures to facilitate the separation of RNA Detergents such as Triton X-100 or SDS, which aid in the solubilization of cell membranes and the release of RNA Reducing Agents: beta-mercaptoethanol to reduce the disulfide bonds in cellular proteins RNase Inhibitors help protect the RNA from degradation by RNases. RNA extraction: phenol-chloroform method  DNA and RNA extraction methods are used to isolate and purify DNA or RNA from biological samples. Key differences DNA: RNA: phenol-chloroform, proteinase K, and other Chemicals and phenol-chloroform-isoamyl alcohol, to preserve chemicals to remove proteins and Reagents the integrity of RNA and prevent its degradation contaminants from the DNA RNA is more sensitive to degradation than DNA Sensitivity More stable due to its single-stranded nature and susceptibility to RNases Both DNA and RNA extraction methods can use column-based purification, organic extraction, or Isolation Techniques magnetic bead-based techniques. However, the specific protocols and reagents may vary to suit the stability and purity requirements of DNA or RNA. DNA is often used for genetic analysis, DNA RNA is used for gene expression studies, real-time Final Applications sequencing, genotyping, and DNA PCR (RT-PCR), microarray analysis, and RNA fingerprinting sequencing (RNA-Seq)  Automated nucleic acid extraction Magnetic beads coated with DNA-binding molecules to selectively capture and separate DNA molecules, making the process highly efficient and suitable for high-throughput applications  การประยุกต์ใช้ในงานทางคลินิก 1. การตรวจวินิจฉัยโรคติดเชื้อ - การสกัด DNA เชื้อวัณโรคจากเสมหะ * ใช้วิธี Solid-phase extraction เพราะรวดเร็ว เหมาะกับงานประจำวัน * ต้องระวังการปนเปื้ อนจาก DNA มนุษย์ - การตรวจเชื้อไวรัสตับอักเสบ 2. การตรวจทางพันธุกรรม * สกัด DNA/RNA จากซีรัม - การตรวจกรองธาลัสซีเมีย * เลือกใช้ชุดสกัดที่เหมาะกับปริมาณไวรัสที่มีน้อย * สกัด DNA จากเลือด EDTA * ต้องการความบริสุทธิ์สูง จึงอาจเลือกวิธี Phenol-chloroform - การตรวจการกลายพันธุ์ของยีนมะเร็ง 3. การศึกษาการแสดงออกของยีน * สกัด DNA จากเนื้อเยื่อ - การติดตามการรักษามะเร็งเม็ดเลือดขาว * ต้องระวังการปนเปื้ อนจากเนื้อเยื่อปกติ * สกัด RNA จากเลือด * ต้องรีบสกัดหลังเก็บตัวอย่างเพราะ RNA เสื่อมสลายเร็ว  แนวทางการเลือกวิธีสกัดที่เหมาะสม พิจารณาชนิดตัวอย่าง พิจารณาทรัพยากร - เลือด -> Solid-phase หรือ Salting-out - ต้องการคุณภาพสูงสุด -> Phenol-chloroform - เนื้ อเยื่อ -> Phenol-chloroform - เวลาน้อย -> Solid-phase - เซลล์เพาะเลี้ยง -> ทุกวิธีใช้ได้ดี - งบประมาณจำกัด -> Salting-out พิจารณาการนำไปใช้ - งานประจำวัน -> Solid-phase - งานวิจัย -> Phenol-chloroform - งานคัดกรอง -> Salting-out  Summary เรียนรู้วิธีการสกัดกรดนิวคลีอิกหลักๆ 3 วิธี แต่ละวิธีมีข้อดีข้อเสียต่างกัน การเลือกใช้ขึ้นอยู่กับ - ชนิดของตัวอย่าง - ความบริสุทธิ์ที่ต้องการ - งบประมาณ - เวลาที่มี - ความชำนาญของผู้ปฏิบัติ