Molecular biology


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Molecular biology

  1. 1. Dr T Balasubramanian<br />Molecular biology<br />drtbalu's otolaryngology online<br />
  2. 2. Introduction<br />These processes govern the behavior of cells<br />Regulates cell functions<br />Regulates cell to cell interactions<br />Regulation of cell turnover<br />drtbalu's otolaryngology online<br />
  3. 3. DNA structure and function<br />Hereditary information are stored in the form of double stranded DNA. These are known as genomes<br />Hydrogen bonds form between complementary pairs of nucleotides, Adenine - Thymine Cytosine – Guanine<br />Nucleotides on each DNA strand are arranged in a linear fashion and are known as codons<br />Each codon codes for a single amino acid<br />There are 64 triplet / codon combinations<br />Commonly encountered amino acids are 20<br />Some amino acids may be represented by different codons<br />Some codons may be considered to be full stop of the genome (stop codons)<br />drtbalu's otolaryngology online<br />
  4. 4. DNA structure and function (contd)<br />99.9% of DNA reside in the cell nucleus (Nuclear genome)<br />Nuclear genome encode 30000 – 350000 genes<br />Mitochondrial genome encode 37 genes<br />Mitochondrial genome studies play an important role in cancer diagnositcs<br />Each DNA molecule is packaged into chromosomes by complex folding of DN A around proteins<br />drtbalu's otolaryngology online<br />
  5. 5. chromosomes<br />DNA molecules are packed inside chromosomes by complex folding around proteins<br />Diploid cells contain 22 pairs of autosomes and one pair of sex chromosome that determines the sex of the individual<br />One of each pair of chromosome is paternal and maternal<br />Each chromosome has two arms separated by a constriction, which is known as the centromere<br />Each chromosome has a distinctive shape, size and banding pattern<br />Long arm of chromosome is indicated by the letter q (for queue) and short arm by the letter p (for petit)<br />Centromeres serves as the point of anchor for the mitotic spindle during cell division <br />drtbalu's otolaryngology online<br />
  6. 6. Telomeres<br />These are specialized sequence of DNA<br />They are supposed to cap the ends of chromosomes<br />They maintain the structural integrity of the chromosome<br />They facilitate complete replication of extreme ends of chromosomes<br />These telomeres are considered to be the key to aging and malignant transformation<br />This protects the genes from degradation / destruction during cell replication during which shortening of chromosome can occur<br />Telomeres which are consumed during cell division are replenished by the enzyme telomerase reverse transcriptase<br />drtbalu's otolaryngology online<br />
  7. 7. Hayflick limit<br />This is the number of times a cell divides before it stops.<br />This stop is due to the loss of Telomeres<br />Each mitosis shortens the Telomeres<br />According to Hayflick human cell divides 40-60 times before entering a phase of senescence<br />Carnosine increases the Hayflick limit by reducing the rate of telomeric shortening<br />drtbalu's otolaryngology online<br />
  8. 8. Telomeres & Cancer<br />Cancer cell require constant production of Telomeres during their cell division<br />The enzyme Telomerase reverse transcriptase is constantly upregulated in these patients<br />The anticancer drug Telomestatin acts against Telomerase enzyme<br />drtbalu's otolaryngology online<br />
  9. 9. Stages of DNA replication<br />Stage of separation of two strands of DNA – DNA polymerase enzyme involved<br />Stage of polymerization: Separated DNA strand acts as a template for this step. Enzyme involved in this stage is DNA polymerase<br />Transcription: This is driven by RNA polymerase. DNA acts as a template. RNA synthesized is complementary to the DNA.<br />Translation: This is a cytoplasmic process. Messenger RNA acts as a template for the new DNA produced<br />DNA proof reading mechanism<br />Stage of DNA repair if errors are present in the replication process. Failure of this repair process is seen in Fanconi’sanaemia, xerodermapigmentosum<br />drtbalu's otolaryngology online<br />
  10. 10. Role of molecular biology in management of head and neck tumors<br />Helps in the identification of malignant tumors<br />Helps in identification of malignant transformation of premalignant lesions<br />Helps in designing the optimal treatment modality<br />DNA is used in the study of molecular biology as it is very stable.<br />drtbalu's otolaryngology online<br />
  11. 11. Reasons for using DNA in molecular biological techniques<br />It is present inside all cells<br />It is fairly stable<br />Can be spliced in to manageable fragments and studied<br />They can easily be cloned / joined<br />Phosphate groups in the DNA confers a negative charge to the DNA enabling electrophoretic migration studies<br />drtbalu's otolaryngology online<br />
  12. 12. Electrophoresis<br />DNA is negatively charged due to the presence of phosphate groups<br />Electrophoresis depends on the migration rate of DNA molecules towards the positive end of the electrode<br />Smaller DNA fragments travel faster towards the positive electrode <br />Larger DNA fragments travel slower <br />Choice of matrix on which DNA fragments move depends on the size of the fragment<br />Commonly used matrix is the polyacrylamide gel<br />drtbalu's otolaryngology online<br />
  13. 13. hybridization<br />This process involves copying of a single strand of DNA<br />Double stranded DNA should be separated before this process could occur<br />Separation of DNA strands can occur when the solution containing DNA is heated<br />The critical temperature at which separation of DNA strands occur is known as Tm<br />When the temperature cools down the two strands join together (annealing)<br />In hybridization radionuleotide tagged probes are used to bind to the single strand of DNA<br />The DNA under study is transferred to nitrocellulose membrane<br />Detection of protein is by using antibodies to specific antigens or by using enzymes that binds to the proteins<br />drtbalu's otolaryngology online<br />
  14. 14. drtbalu's otolaryngology online<br />
  15. 15. cytogenetics<br />This is a study of chromosomal abnormalities and rearrangements<br />Plays a major role in prenatal diagnosis of Down’s syndrome and other genetic abnormalities<br />TheodareBoveri postulated that cancer arose from chromosomal aberrations<br />Chronic myeloid leukemia occurs due to translocation of chromosome<br />Cytogenetic studies play a vital role in the diagnosis of reticuloendothelialmalginancies<br />drtbalu's otolaryngology online<br />
  16. 16. FISH<br />Fluorescent In situ hybridization is a vital study in molecular genetics<br />This is used to localize / detect the presence of DNA sequences on chromosomes<br />In this test fluorescent probes are used <br />These probes will bind to those portions in the chromosomes showing DNA similarity<br />Fluorescent microscopes are used to identify the chromosome bound probes<br />drtbalu's otolaryngology online<br />
  17. 17. FISH uses<br />Identifying DNA sequences<br />Predicting genetic disorders / malignancies<br />Counseling<br />Used to detect specific messenger RNAs in the tissue studied<br />drtbalu's otolaryngology online<br />
  18. 18. Fish stages<br />Construction of Probe<br />Tagging probes with flurophores<br />Interphase chromosomal preparation is produced<br />Blocking of repetitive DNA sequences<br />Incubation of probe with chromosome DNA<br />Repeated washing to remove unhybridized / partially hybridized probes<br />Examination under florescent microscope<br />drtbalu's otolaryngology online<br />
  19. 19. drtbalu's otolaryngology online<br />
  20. 20. Polymerase chain reaction<br />This test had the most dramatic influence on molecular genetics<br />The amount of genetic material needed for accurate analysis is miniscule when compared to FISH<br />PCR manages to achieve faithful and exponential amplification of the genetic material under study<br />drtbalu's otolaryngology online<br />
  21. 21. Pcr steps<br />Denaturation of double stranded D NA<br />Annealing of oligonucleotide primers<br />Extension of new strands by polymerase<br />This cycle is repeated.<br />28 cycles generates about 67 million copies of DNA<br />drtbalu's otolaryngology online<br />
  22. 22. Thank you<br />drtbalu's otolaryngology online<br />