DNA, RNA Analysis PDF

DNA, RNA Analysis by Prof. Nagwa Abo El-Maali DNA Facts Chromosomes are made of DNA Deoxyribonucleic Acid (DNA) Molecule that stores genetic information in cells Copies itself exactly for new cells DNA-Deoxyribonucleic Acid DNA contains the in...

DNA, RNA Analysis by Prof. Nagwa Abo El-Maali DNA Facts Chromosomes are made of DNA Deoxyribonucleic Acid (DNA) Molecule that stores genetic information in cells Copies itself exactly for new cells DNA-Deoxyribonucleic Acid DNA contains the instructions for making proteins within the cell Why is the Study of DNA Important? It’s essential to all life on earth Medical Benefits—disease detection, treatment, prevention Development of Crops Forensics DNA Structure  DNA is a polymer (composed of repeating subunits called nucleotides)  2 long strands  Each a chain of nucleotides Nucleotides  Consists of…  Phosphate  Carbon sugar (deoxyribose)  Nitrogen base Types of Nitrogenous Bases DNA Strand  A = adenine  Each nucleotide bonds to the next one to  T = thymine form a strand.  C = cytosine  The two strands twist around a  G = guanine central axis to form a double helix.  Sides of the ladder alternate phosphate and sugar (deoxyribose)  Rings are held together by Hydrogen bonds Base Pair Rule Nitrogenous Bases  Adenine can bond Those 4 bases (ATCG) have endless only with Thymine combinations  A-T or T-A (2 H Just like the letters bonds) of the alphabet can combine to make an  Cytosine can bond infinite number of words. only with Guanine The two strands are said  C-G or G-C (3 H to be complimentary bonds) That means that if  This is called the you have GAATAC on one side you will BASE PAIR RULE have _ _ _ _ _ _ on the other.  Translation DNA is in the nucleus To make proteins, DNA must get its instructions to the ribosomes who make proteins. To transport its instructions, it uses Messenger RNA (mRNA)  RNA Ribonucleic Acid Consists only of one strand of nucleotides Has ribose (a 5C sugar) NOT deoxyribose Has uracil (U) as a nitrogenous base NOT thymine  DNA by the Numbers Each cell has about 3 meters of DNA. The average human has 300 trillion cells. The average human has enough DNA to go from the earth to the sun more than 400 times. DNA has a diameter of only 0.000000002 meters (2 x10-9m) The earth is 150 billion meters or 93 million miles from the sun. DNA and RNA Extraction To study or manipulate nucleic acids, the DNA or RNA must first be isolated or extracted from the cells, this can be done through various techniques. Most nucleic acid extraction techniques involve steps to break open the cell and use enzymatic reactions to destroy all macromolecules that are not desired (such as degradation of unwanted molecules and separation from the DNA sample). Cells are broken using a lysis buffer (a solution that is mostly a detergent); lysis means “to split.” These enzymes break apart lipid molecules in the membranes of the cell and the nucleus. Macromolecules are inactivated using enzymes such as proteases that break down proteins, and ribonucleases (RNAses) that break down RNA. The DNA is then precipitated using alcohol. Human genomic DNA is usually visible as a gelatinous, white mass. Samples can be stored at –80°C for years. Figure: DNA Extraction: This diagram shows the basic method used for extraction of DNA. RNA analysis is performed to study gene expression patterns in cells. RNA is naturally very unstable because RNAses are commonly present in nature and very difficult to inactivate. Similar to DNA, RNA extraction involves the use of various buffers and enzymes to inactivate macromolecules and preserve the RNA. Nucleic acid detection and quantification methods Nucleic acid is purified from cells as part of an ever- growing array of molecular biology methods, including sequencing and gene editing. Before they are used in downstream applications, nucleic acids are detected and quantitated using UV or fluorescence spectrophotometry. Traditionally measured individually in cuvettes, sample analysis is now routinely performed in microplates. Molecular Devices provides a complete workflow solution for nucleic acid detection, quantitation, and analysis. The quantitation and analysis of nucleic acids: DNA/RNA absorbance measurements Fluorometric quantitation of nucleic acids SNP genotyping DNA/RNA absorbance measurements The absorbance of a DNA sample measured at 260 nm on a spectrophotometer or microplate reader can be used to calculate its concentration. Absorbance quantitation of DNA works on samples ranging from about 0.25 μg/mL to about 125 μg/mL in a microplate format. Fluorometric quantitation of nucleic acids Quantitation of DNA is a critical step in molecular biology requiring accuracy, reliability, and the use of increasingly smaller sample volumes for applications such as next- generation sequencing. Compared to spectrophotometric DNA quantitation, the fluorometric method provides key advantages such as significantly increased sensitivity, high selectivity for double- stranded DNA (dsDNA) over single-stranded DNA (ssDNA) or RNA, and improved contaminant tolerance (protein and carbohydrate molecules). Single nucleotide polymorphisms (SNP) genotyping Genotyping is a process for analyzing genetic differences among individuals by examining their DNA sequences. SNPs are one of the most common types of genetic variation, consisting of a single nucleotide mutation at a specific locus. SNP genotyping has proven very useful in identifying disease-related mutations in various species, and as a result many techniques for SNP detection have been developed. Electrophoresis Denaturation: the change of folding structure of a protein (and thus of physical properties) caused by heating, changes in pH, or exposure to certain chemicals Electrophoresis: a method for the separation and analysis of large molecules, such as proteins or nucleic acids, by migrating a colloidal solution of them through a gel under the influence of an electric field Polymerase chain reaction: a technique in molecular biology for creating multiple copies of DNA from a sample  The first step to study or work with nucleic acids includes the isolation or extraction of DNA or RNA from cells. Gel electrophoresis depends on the negatively-charged ions present on nucleic acids at neutral or basic pH to separate molecules on the basis of size. Specific regions of DNA can be amplified through the use of polymerase chain reaction for further analysis. Southern blotting involves the transfer of DNA to a nylon membrane, while northern blotting is the transfer of RNA to a nylon membrane; these techniques allow samples to be probed for the presence of certain sequences. Basic Techniques to Manipulate Genetic Material (DNA and RNA) To understand the basic techniques used to work with nucleic acids, remember that nucleic acids are macromolecules made of nucleotides (a sugar, a phosphate, and a nitrogenous base) linked by phosphodiester bonds. The phosphate groups on these molecules each have a net negative charge. An entire set of DNA molecules in the nucleus is called the genome. DNA has two complementary strands linked by hydrogen bonds between the paired bases. The two strands can be separated by exposure to high temperatures (DNA denaturation) and can be reannealed by cooling. The DNA can be replicated by the DNA polymerase enzyme. Unlike DNA, which is located in the nucleus of eukaryotic cells, RNA molecules leave the nucleus. The most common type of RNA that is analyzed is the messenger RNA (mRNA) because it represents the protein -coding genes that are actively expressed. Gel Electrophoresis Because nucleic acids are negatively-charged ions at neutral or basic pH in an aqueous environment, they can be mobilized by an electric field. Gel electrophoresis is a technique used to separate molecules on the basis of size using this charge and may be separated as whole chromosomes or fragments. The nucleic acids are loaded into a slot near the negative electrode of a porous gel matrix and pulled toward the positive electrode at the opposite end of the gel. Smaller molecules Nucleic acids in a gel move through the There are matrix can be observed pores in the gel molecular-weight using various fluorescent faster than larger standard samples or colored dyes. Distinct molecules; this that can be run nucleic acid fragments difference in the rate alongside the appear as bands at of migration molecules to specific distances from separates the provide a size the top of the gel (the fragments on the comparison. negative electrode end) basis of size. on the basis of their size. Figure: Gel Electrophoresis: Shown are DNA fragments from seven samples run on a gel, stained with a fluorescent dye, and viewed under UV light. Amplification of Nucleic Acid Fragments by Polymerase Chain Reaction Polymerase chain reaction (PCR) is a technique used to amplify specific regions of DNA for further analysis. PCR is used for many purposes in laboratories, such as: Cloning of gene fragments to analyze genetic diseases, Identification of contaminant foreign DNA in a sample, and Amplification of DNA for sequencing. Determination of paternity and detection of genetic diseases. PCR Amplification: Polymerase chain reaction, or PCR, is used to amplify a specific sequence of DNA. Primers—short pieces of DNA complementary to each end of the target sequence— are combined with genomic DNA, Taq polymerase, and deoxynucleotides. Taq polymerase is a DNA polymerase isolated from the thermostable bacterium Thermus aquaticus that is able to withstand the high temperatures used in PCR. (Thermus aquaticus grows in the Lower Geyser Basin of Yellowstone National Park).  Reverse transcriptase PCR (RT-PCR) is similar to PCR, but cDNA is made from an RNA template before PCR begins. DNA fragments can also be amplified from an RNA template in a process called reverse transcriptase PCR (RT-PCR). The first step is to recreate the original DNA template strand (called cDNA) by applying DNA nucleotides to the mRNA. This process is called reverse transcription. This requires the presence of an enzyme called reverse transcriptase. After the cDNA is made, regular PCR can be used to amplify it.  DNA fragments can also be amplified from an RNA template in a process called reverse transcriptase PCR (RT-PCR). The first step is to recreate the original DNA template strand (called cDNA) by applying DNA nucleotides to the mRNA. This process is called reverse transcription. This requires the presence of an enzyme called reverse transcriptase. After the cDNA is made, regular PCR can be used to amplify it. Hybridization, Southern Blotting, and Northern Blotting Nucleic acid samples, such as fragmented genomic DNA and RNA extracts, can be probed for the presence of certain sequences. Short DNA fragments called probes are designed and labeled with radioactive or fluorescent dyes to aid detection. Gel electrophoresis separates the nucleic acid fragments according to their size. The fragments in the gel are then transferred onto a nylon membrane in a procedure called blotting.