molecular biology

DRTBALU'S OTOLARYNGOLOGY ONLINE

Dr T Balasubramanian

MOLECULAR BIOLOGY


INTRODUCTION• These processes govern the behavior of cells

• Regulates cell functions

• Regulates cell to cell interactions

• Regulation of cell turnover


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)

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There are more triplet codons i.e. 64 than the commonly encountered aminoacids (20 in number)


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


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


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


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


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


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’s anaemia, xeroderma pigmentosum


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.


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


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


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


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

• Theodare Boveri 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 reticuloendothelial malginancies


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


FISH USES• Identifying DNA sequences

• Predicting genetic disorders / malignancies

• Counseling

• Used to detect specific messenger RNAs in the tissue studied


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


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


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


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molecular biology

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