Molecular biology
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Molecular biology

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Molecular biology Molecular biology Presentation Transcript

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