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    MTPC 140: Molecular Biology and Diagnostics

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    Valdez Francis Zaccheau
    This document provides an overview of molecular biology and diagnostic techniques taught in MTPC 140. It discusses the history of key discoveries in molecular biology including the identification of DNA as the genetic material. The central dogma of molecular biology involving DNA replication, transcription, and translation is explained. Common nucleic acid extraction methods from samples, amplification techniques, and detection methods used in diagnostics are also summarized.

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    MTPC 140: Molecular Biology and Diagnostics

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    Valdez Francis Zaccheau
    78 views40 pages
    This document provides an overview of molecular biology and diagnostic techniques taught in MTPC 140. It discusses the history of key discoveries in molecular biology including the identification of DNA as the genetic material. The central dogma of molecular biology involving DNA replication, transcription, and translation is explained. Common nucleic acid extraction methods from samples, amplification techniques, and detection methods used in diagnostics are also summarized.

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    Reinforcement+in+MTPC+140

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    This document provides an overview of molecular biology and diagnostic techniques taught in MTPC 140. It discusses the history of key discoveries in molecular biology including the identification of DNA as the genetic material. The central dogma of molecular biology involving DNA replication, transcription, and translation is explained. Common nucleic acid extraction methods from samples, amplification techniques, and detection methods used in diagnostics are also summarized.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    78 views40 pages

    MTPC 140: Molecular Biology and Diagnostics

    Uploaded by

    Valdez Francis Zaccheau
    This document provides an overview of molecular biology and diagnostic techniques taught in MTPC 140. It discusses the history of key discoveries in molecular biology including the identification of DNA as the genetic material. The central dogma of molecular biology involving DNA replication, transcription, and translation is explained. Common nucleic acid extraction methods from samples, amplification techniques, and detection methods used in diagnostics are also summarized.

    Copyright:

    © All Rights Reserved
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    Download as pdf or txt
    of 40

    MTPC 140: Molecular Biology

    and Diagnostics
    MAVERICK V. SUSTIGUER, RMT
    Senior Instructor
    Medical Technology Department
    Notre Dame of Marbel University
    Investigator Contribution
    Friedrich Miescher Isolated nuclein in white blood cell nuclei
    Transferred killing ability between types of bacteria
    Frederick Griffith S. pneumoniae: Type R to Type S
    BACTERIAL TRANSFORMATION
    Oswald Avery
    Discovered that DNA transmits killing ability in bacteria
    Colin McLeod
    BACTERIAL TRANSFORMATION-uptake of FREE DNA in the environment
    Maclyn McCarty

    Determined that the part of a virus that infects and replicates is its nucleic acid and not its
    Alfred Hershey
    protein
    Martha Chase
    BACTERIAL TRANSDUCTION: Bacteriophage
    Phoebus Levene Discovered DNA components, proportions and positions
    Erwin Chargaff • Sugar (deoxyribose and ribose)
    Maurice Wilkins • A=T; G=C
    Rosalind Franklin • Photo 51
    James Watson
    Elucidated DNA’s three-dimensional structure
    Francis Crick
    James Watson Had his genome sequenced
    CENTRAL DOGMA: transfer of important information in biological system

    • Nucleus
    • Nucleus • Promoter (TATA box), Transcription factors,
    •
    •
    S phase (interphase) Replication
    Semiconservative replication Two dsDNA
    DNA Elongation factors, RNA polymerase
    • Initiation → Elongation → Termination
    • 1 dsDNA → 2dsDNA • dsDNA → transcribed ssRNA → mRNA
    • Helicase, Primase, DNA Transcription • Capping → Polyadenylation → Splicing
    polymerase III, DNA mRNA
    polymerase I, DNA Ligase
    RNA
    • Cytoplasm (ribosomes)
    Translation
    • Initiation → Elongation → Termination Polypeptide
    • mRNA, tRNA, ribozyme, ribosome
    (small and large subunit)
    • P site, A site, E site
    • Codon, anticodon and Amino acid
    Protein
    • Genetic code → Proteins

    10/04/2021 Maverick V. Sustiguer, RMT 9


    Replication
    • Nucleus
    • S phase (interphase) *resting/nondividing
    Gap 1 – S phase (8 hours) – Gap 2
    • Semiconservative replication: 1 dsNA → 2 dsDNA (one
    original and one newly synthesized strand)
    Three postulates: Conservative, Semiconservative, Dispersive
    (𝑁𝑁 14 / 𝑁𝑁 15 )
    • Unidirectional (5’-3’): Leading strand (continuous) and
    Lagging strand (discontinuous) *Okazaki fragments
    • 1 dsDNA→ 2dsDNA (antiparallel)
    • Enzymes
    1. Helicase: disrupts hydrogen bonds
    2. Primase: attachment of RNA primer
    3. DNA polymerase III: attachment of free nucleotides
    (dNTPs)
    4. DNA polymerase I: proofreading of mismatches
    5. DNA ligase: phosphodiester bond
    10/04/2021 Maverick V. Sustiguer, RMT 10
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    Transcription
    • Nucleus
    • Interphase *resting/nondividing
    • dsDNA → transcribed ssRNA (hnRNA) → mRNA (codon)
    • Unidirectional (5’-3’) replication
    • dsDNA (antisense and sense strand) *Uracil replaces thymine
    • Initiation → Elongation → Termination
    • Locate promoter → Attachment of TATA-binding protein → Attachment of TF → Binding of
    RNA polymerase (TATA box) →Attachment of EF → Unwinding of dsDNA → Transcription of
    DNA strand → Terminator (Hairpin/Loop structure) → Release
    • mRNA processing hnRNA A A UG U AA G AA
    1. Capping: 7-methylguanosine
    2. Polyadenylation: 250 adenine residues
    A U G mRNA
    3. Splicing: spliceosome (introns and exons)
    10/04/2021 Maverick V. Sustiguer, RMT 14
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    10/04/2021 Maverick V. Sustiguer, RMT 16
    10/04/2021 Maverick V. Sustiguer, RMT 17
    10/04/2021 Maverick V. Sustiguer, RMT 18
    10/04/2021 Maverick V. Sustiguer, RMT 19
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    10/04/2021 Maverick V. Sustiguer, RMT 21
    Translation
    • Decoding of the genetic code to produce proteins
    • Cytoplasm (Ribosomes)
    • Gap 1 and Gap 2 (Interphase) *resting/nondividing
    Gap 1 – S phase – Gap 2
    • tRNA (anticodon and AA), mRNA (codon), rRNA, ribosome (small and large subunit) *nucleolus
    • Initiation → Elongation → Termination
    • Small subunit + IF-3 + 5’ of mRNA → IF-2 + GTP + tRNA → Association of large subunit (release of
    GDP, phosphate, IF) → P site → E site * starting codon (A site → P site → E site * other codons)
    → Stop codons: UAA, UAG, UGA → Release of polypeptide → Disassembly of ribosome

    AUG GUU GCU GAU GGU UGU UGA

    Met-Val-Ala-Asp-Gly-Cys
    10/04/2021 Maverick V. Sustiguer, RMT 22
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    Area A Area B

    Area D Area C
    Area A Area B

    Area D Area C
    Nucleic Acid Nucleic Acid Nucleic Acid
    Sample Collection
    Extraction Amplification Detection

    Whole blood, plasma, bone marrow, urine,


    feces, dried blood spot, body fluids, amniotic
    fluid, CVS, buccal cells, saliva, tissue, bone,
    hair, nails

    COMMON EXTRACTION METHODS


    • Liquid-phase extraction (large sample volumes)
    A. Organic extraction: differential solubility
    • Phenol-chloroform
    • Hydrophobic portion – lipids and cell debris (bottom)
    • Hydrophilic portion – DNA (top)
    B. Inorganic extraction: high concentration of salts at ↓ pH
    DNA collected in the upper phase and precipitated w/ isopropyl alcohol
    •
    • Solid-phase extraction (commonly used)
    A. Column-based extraction: selective binding of nucleic acid to solid matrix in a column
    B. Magnetic bead extraction: small particles with paramagnetic core coated by a surface that binds
    nucleic acid
    10/04/2021 Maverick V. Sustiguer, RMT 33
    ORGANIC EXTRACTION METHOD

    Aqueous Nucleic acid


    phase recovery by
    Phenol Centrifugation
    alcohol
    chloroform precipitation
    Organic
    phase

    Cell lysate Biphasic emulsion Phase separation Organic extraction of DNA and RNA are very similar.
     DNA extraction: pH 7-8
     RNA extraction: pH 4-6

    10/04/2021 Maverick V. Sustiguer, RMT 34


    INORGANIC EXTRACTION METHOD

    DNA in
    solution
    Nucleic acid
    recovery by
    High salt Centrifugation
    solution alcohol
    precipitation
    Protein pellet

    Cell lysate Cell-lysate salt Protein


    mixture precipitation
    10/04/2021 Maverick V. Sustiguer, RMT 35
    MAGNETIC-BASED EXTRACTION
    METHOD

    Addition of Addition
    Addition of proteinase of Nucleic acid Elution
    Sample Washing Purified
    lysis buffer K magnetic magnetic and nucleic acid
    10/04/2021 beads
    Maverick V. separation
    Sustiguer, RMT transfer 37
    Transfer Addition of
    Cell ethanol/isoprop Storage/
    aqueous
    suspension anol and salt processing
    phase solution
    Aqueous phase
    (top) and Addition of
    Lysis Centrifuge
    Organic phase buffer/water
    (bottom)

    Cell lysate
    DNA pellet + Dry the DNA
    with Centrifuge
    70% ethanol pellet
    contaminants

    Addition of Biphasic Discard


    phenol:chlorofor Centrifuge
    m emulsion supernatant
    10/04/2021 Maverick V. Sustiguer, RMT 38
    Assessment of Nucleic Acid Yield
    Method Principle Interpretation
    DNA 𝜇𝜇g/mL: A260 X 50 X DF*
    Nucleic acids RNA 𝜇𝜇g/mL: A260 X 40 X DF*
    Spectrophotometry absorb UV light at
    260 nm Absorbance is directly proportional to the
    concentration of the nucleic acid.

    Intensity of band is Brighter bands = higher yield


    directly proportional *plasmid DNA: bright, moderate mobility single
    to nucleic acid band
    Gel electrophoresis
    concentration *HW genomic DNA: bright band with low mobility

    Ethidium bromide and SYBR Green I: DNA


    Nucleic acids → Anode Ethidium bromide and SYBR Green II: RNA
    10/04/2021 Maverick V. Sustiguer, RMT 39
    Assessment of Nucleic Acid Quality
    DNA A260/A280 ratio:
    • 1.6-2.0: good quality
    • 1.6-1.9: minimal contamination
    • 1.8-2.0: clinical specimens
    Nucleic acids absorb UV light at 260 nm
    Spectrophotometry • 2.0: pure DNA extract
    Proteins absorb light at 280 nm • <1.6: protein contamination
    • >2.0: contamination with RNA
    RNA A260/A280 ratio
    • ≥2.0: good quality
    Position of the band is related to molecular DNA:
    weight High MW fragments: good quality
    Gel electrophoresis Fragments with lower MW migrate furthest RNA 28S/18S ratio:
    from point of application 2: good quality
    ≤2.0: smaller fragments

    10/04/2021 Maverick V. Sustiguer, RMT 40

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