Analysis Of Nucleic Acids - PDF

Analysis of nucleic acids Zsolt Fábián M.D., Ph.D., Dr. Habil.  Analysis of nucleic acids Sickle Cell Disease A 3.5-year-old girl has been referred to Shafa Hospital with sever...

Analysis of nucleic acids Zsolt Fábián M.D., Ph.D., Dr. Habil.  Analysis of nucleic acids Sickle Cell Disease A 3.5-year-old girl has been referred to Shafa Hospital with severe anemia, thrombocytopenia, leukocytosis and elevated serum lactate dehydrogenase (LDH) enzyme. Her parents assigned fever, cough, pallor, weakness and tachypnea. On examination, she had fever, anemia, hepatosplenomegaly and dactylitis (a). Peripheral blood smear revealed sickling of red blood cells (b) leading to the tentative diagnosed of sickle cell anemia. a b M Pedram, et al., Iran Red Crescent Med J. 2012 Mar; 14(3): 184–185.  Analysis of nucleic acids Sickle Cell Disease ~1 in 365 African Americans ~1 in 16,300 Hispanic Americans Autosomal Recessive Caused by an A → T transversion in the HBB encoding β-globin Amino Acid 6: glutamic acid (E) → valine (V). E6V – Results in abnormal hemoglobin (HbS) Normal adult hemoglobin is HbA Clinical Presentation: – Presents within first 2 years – Severe hemolysis – Crises lasting ~5-7 days – Hemolytic anemia, failure to thrive, splenomegaly – Painful swelling of hands/feet (occlusion of capillaries) – Repeated infections Infection is a major cause of death Treatments – Hydroxyurea – Supportive including O2, transfusion, penicillin  Analysis of nucleic acids Agarose gelelectrophoresis RNA and DNA molecules can be separated by size using gel electrophoresis. RNA and DNA are negatively charged so will move toward the positive electrode when placed in a electric field (a). A gel matrix made of agarose or polyacrylamide slows the movement of the molecules (b). Larger molecules move more slowly through the gel than smaller molecules (c).  Methods of molecular cell biology I Agarose gelelectrophoresis RNA and DNA molecules can be separated by size using gel electrophoresis. RNA and DNA are negatively charged so will move toward the positive electrode when placed in a electric field. A gel matrix made of agarose or polyacrylamide slows the movement of the molecules. Larger molecules move more slowly through the gel than smaller molecules. Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Analysis of nucleic acids Restriction endonuclease cleavage DNA Nucleases Enzymes that digest DNA by cleaving the phosphodiester bonds between nucleotides Exonucleases digest from a free end Endonucleases digest at an internal site Restriction Endonucleases Recognize a specific DNA sequence and cleave a phosphodiester bond in both strands within that sequence Highly specific  Always cleaves at the same DNA sequence -> restriction site  Only cleaves within that sequence Restriction Sites Often 4-8 bp in length 5’ GGATCC 3’ Often DNA palindromes  The same 5’ to 3’ sequence on complimentary strands 3’ CCTAGG 5’  Analysis of nucleic acids Restriction endonuclease cleavage Restriction enzymes cut DNA at specific palindromic sequences Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Analysis of nucleic acids Restriction endonuclease cleavage Digestion of a DNA sample with a restriction enzyme Sample has been separated by gel electrophoresis Gel has been counterstained with a DNA-specific red fluorescent dye  Analysis of nucleic acids Nucleic aids – Hybridization Hybridization Annealing of a single-strand of DNA to a complementary strand of a different DNA molecule Used in many molecular techniques – Allows detection of DNA molecules of a specific sequence in complex mixtures 100% complementarity is not necessary. By altering conditions, we can control the degree to which base-pair mismatches are tolerated Stringency = the ‘degree to which mismatches are tolerated’ in a hybridization reaction  Analysis of nucleic acids Molecular hybridization Target: A complex mixture of nucleic acid fragments being tested. Probe: A nucleic acid fragment of known sequence. Complimentary to the sequence to be detected. Labeled to allow detection.  Analysis of nucleic acids Molecular hybridization - Southern botting A specific DNA sequence within a DNA sample can be identified with a Southern blot analysis DNA is cut with specific restriction endonucleases and the different fragments separated by size with gel electrophoresis The DNA fragments are denatured with an alkali to expose the bases and are transferred to a membrane The membrane is then probed with a radioactively labeled single stranded DNA molecule that has a complementary sequence to the target DNA The probe hybridizes with any DNA fragments containing the target sequence. These fragments can be identified from an autoradiogram Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Analysis of nucleic acids Restriction fragment length polymorphism (RFLP) Genomic DNA β-Globin gene EcoRI EcoRI EcoRI EcoRI EcoRI Healthy X Sickle cell anemia EcoRI digestion EcoRI digestion Southern blotting for β-globin gene sequences Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Analysis of nucleic acids Restriction fragment length polymorphism (RFLP) Maternal allele Paternal allele EcoRI EcoRI EcoRI EcoRI EcoRI X Sickle cell anemia EcoRI digestion EcoRI digestion Southern blotting for β-globin gene sequences Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Analysis of nucleic acids Large deletions detected by southern blotting * * * * 1 2 3 4 5 6 7 8 9 C Southern blot analysis of Exon 3 Exons 1,2 LMX1B in Nail-Patella Syndrome (NPS) patients Autosomal dominant Exons 4-6 Identify individuals heterozygous for gene Exons 7,8 deletions  Less target DNA results in a COL10A1 weaker signal  Analysis of nucleic acids Restriction fragment length polymorphism (RFLP) EcoRI site EcoRI site Genomic DNA Alu sequences alleles Alu sequences maternal Alu sequences Alu sequences paternal Alu sequences Southern blotting for Alu sequences Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  Analysis of nucleic acids DNA fingerprinting using minisatellites Mother Child 1 Child 2 Child 3 Child 4 Father Fragment length  Analysis of nucleic acids Blotting methodologies Gel electrophoresis to separate target samples by size Southern Blotting – DNA target separated by gel electrophoresis – Transferred onto membrane – Detection of the DNA target using complementary DNA probe Northern blotting – RNA target separated by gel electrophoresis – transferred onto membrane – Detection of the RNA target using complementary DNA probe Western blotting – Protein target separated by gel electrophoresis – Transferred onto membrane – Detection of the protein target using antibodies against protein  Analysis of nucleic acids Key points 1. In gel electrophoresis, linear nucleic acids migrate on the basis of their length. After separation, the smaller the molecule, the close it will be to the anode. 2. Restriction endonucleases cleave double-stranded DNA at specific sequences. They usually recognize palindromic sequences and produce sticky or blunt ended restriction fragments. 3. Nucleic acid hybridization assays use a labelled probe specific to a specific target sequence within a complex mixture of nucleic acids. 4. In blotting assays, target molecules are separated by gel electrophoresis, immobilized on a solid support and hybridized using a labelled probe. 5. Southern blotting is used for detecting specific DNA molecules, northern blots are used for detecting specific RNA molecules, and western blots are used for detecting specific proteins.  Analysis of nucleic acids Key points 6. Restriction Fragment Length Polymorphisms are DNA sequence variants that result in an alteration of a genomic DNA restriction fragment. These are often caused by Single Nucleotide Polymorphisms (SNPs). 7. Northern blots are most often used for determining the level of expression of a gene in various tissues or individuals. 8. Sickle cell disease is caused by a specific E6V mutation in the HBB gene encoding β-globin. It has autosomal recessive inheritance. It presents with crises, featuring severe hemolytic anemia, splenomegaly, painful swelling of the hands and feet and repeated infections. 9. Sickle cell trait occurs in heterozygous carriers of the sickle cell mutation. It is largely asymptomatic but rare crises can occur in conditions of severe oxidative stress.