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    Molecular Techniques

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    ANA
    This document describes several important molecular techniques used in research laboratories, including DNA cloning, polymerase chain reaction (PCR), gel electrophoresis, hybridization, and array-based expression profiling. DNA cloning involves inserting a DNA fragment into a plasmid vector to generate multiple copies. PCR amplifies specific DNA regions using primers and DNA polymerase. Gel electrophoresis separates DNA fragments by size on an agarose or polyacrylamide gel. Hybridization techniques like Southern and Northern blots detect DNA and RNA through probe hybridization. Array-based methods analyze gene expression by hybridizing targets to microarrays on glass slides or chips.

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    © All Rights Reserved
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    Molecular Techniques

    Uploaded by

    ANA
    48 views30 pages
    This document describes several important molecular techniques used in research laboratories, including DNA cloning, polymerase chain reaction (PCR), gel electrophoresis, hybridization, and array-based expression profiling. DNA cloning involves inserting a DNA fragment into a plasmid vector to generate multiple copies. PCR amplifies specific DNA regions using primers and DNA polymerase. Gel electrophoresis separates DNA fragments by size on an agarose or polyacrylamide gel. Hybridization techniques like Southern and Northern blots detect DNA and RNA through probe hybridization. Array-based methods analyze gene expression by hybridizing targets to microarrays on glass slides or chips.

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    This document describes several important molecular techniques used in research laboratories, including DNA cloning, polymerase chain reaction (PCR), gel electrophoresis, hybridization, and array-based expression profiling. DNA cloning involves inserting a DNA fragment into a plasmid vector to generate multiple copies. PCR amplifies specific DNA regions using primers and DNA polymerase. Gel electrophoresis separates DNA fragments by size on an agarose or polyacrylamide gel. Hybridization techniques like Southern and Northern blots detect DNA and RNA through probe hybridization. Array-based methods analyze gene expression by hybridizing targets to microarrays on glass slides or chips.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    48 views30 pages

    Molecular Techniques

    Uploaded by

    ANA
    This document describes several important molecular techniques used in research laboratories, including DNA cloning, polymerase chain reaction (PCR), gel electrophoresis, hybridization, and array-based expression profiling. DNA cloning involves inserting a DNA fragment into a plasmid vector to generate multiple copies. PCR amplifies specific DNA regions using primers and DNA polymerase. Gel electrophoresis separates DNA fragments by size on an agarose or polyacrylamide gel. Hybridization techniques like Southern and Northern blots detect DNA and RNA through probe hybridization. Array-based methods analyze gene expression by hybridizing targets to microarrays on glass slides or chips.

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    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 30

    Molecular Techniques:

    Tools for Success


    Jason Rosenzweig, PhD

    From Bench to Bedside - Molecular Techniques: Tools for Success


    Outline
    • Introduction
    • DNA Cloning
    • Polymerase Chain Reaction (PCR)
    • Electrophoresis
    • Hybridization
    • Array-based expression profiling

    From Bench to Bedside - Molecular Techniques: Tools for Success


    Introduction

    From Bench to Bedside - Molecular Techniques: Tools for Success


    DNA

    Alberts, et al. 2002

    From Bench to Bedside - Molecular Techniques: Tools for Success


    RNA

    Alberts, et al. 2002

    From Bench to Bedside - Molecular Techniques: Tools for Success


    Proteins

    Alberts, et al. 2002


    From Bench to Bedside - Molecular Techniques: Tools for Success
    DNA Cloning

    From Bench to Bedside - Molecular Techniques: Tools for Success


    DNA Cloning
    DNA fragment Plasmid

    DNA Isolation

    Cell
    Bacteria Culture

    From Bench to Bedside - Molecular Techniques: Tools for Success


    DNA Cloning - Vector Construction
    Expression Systems:
    Promoter
    •Bacterial
    •Yeast
    •Baculovirus (Insect)
    •Plant Cloning
    •Mammalian Selection marker/ site
    •Cell-free Resistance gene

    Replication
    Origin

    From Bench to Bedside - Molecular Techniques: Tools for Success


    DNA Cloning - Restriction Nucleases
    Type II Restriction nucleases:
    • Recognize specific DNA sequences

    • Usually four to eight bases and palindromic

    • Cut the DNA – can be blunt or “sticky”

    Alberts, et al. 2002


    From Bench to Bedside - Molecular Techniques: Tools for Success
    DNA Cloning - Ligation
    Sticky Ends

    Blunt Ends

    Alberts, et al. 2002


    From Bench to Bedside - Molecular Techniques: Tools for Success
    Polymerase Chain Reaction

    From Bench to Bedside - Molecular Techniques: Tools for Success


    Polymerase Chain Reaction
    DNA template and primers
    Heat to denature DNA

    Cool to anneal primers

    DNA polymerase uses original DNA


    as a template to make a copy

    Resulting DNA is one strand of original


    template and one newly synthesized strand

    From Bench to Bedside - Molecular Techniques: Tools for Success


    Quantitative PCR (qPCR)
    Dye-based (SYBR) Probe-based (TaqMan)
    DNA-binding dye fluoresces when Probe labeled with a fluorophore and a
    bound to double stranded DNA quencher.
    Probe binds a specific sequence between
    the primers
    Probe is digested during DNA synthesis,
    releasing fluorophore from quencher.

    PCR

    PCR

    From Bench to Bedside - Molecular Techniques: Tools for Success


    qPCR

    From Bench to Bedside - Molecular Techniques: Tools for Success


    Reverse Transcriptase PCR (RT-PCR)
    Quantifying gene expression

    RNA

    Reverse transcriptase, DNA primers

    complementary DNA (cDNA)


    RNA

    cDNA used as template for qPCR

    From Bench to Bedside - Molecular Techniques: Tools for Success


    Gel Electrophoresis

    From Bench to Bedside - Molecular Techniques: Tools for Success


    Gel Electrophoresis

    Electricity + Time

    From Bench to Bedside - Molecular Techniques: Tools for Success


    Gel Electrophoresis – Two Dimensions
    Isoelectric Focusing
    4 8 6 5 4 4 4 5
    6 5 8 5 ` 8 6
    7 7 6 7 7
    8

    Alberts, et al. 2002

    From Bench to Bedside - Molecular Techniques: Tools for Success


    Hybridization Techniques

    From Bench to Bedside - Molecular Techniques: Tools for Success


    Hybridization Techniques

    • Southern Blot (DNA) – labeled, single-stranded


    nucleic acid probes to detect DNA

    • Northern Blot (RNA) – labeled, single-stranded


    nucleic acid probes to detect RNA

    • Western Blot (Protein) – labeled probes to detect


    specific protein (primary and secondary antibodies)

    • Enzyme-Linked Immunosorbent Assay (ELISA) -

    From Bench to Bedside - Molecular Techniques: Tools for Success


    Hybridization – Southern and Northern

    Alberts, et al. 2002


    From Bench to Bedside - Molecular Techniques: Tools for Success
    Hybridization – Western Blot
    Transfer proteins from gel to membrane Hybridization

    Visualization

    Winston et al. 2008

    From Bench to Bedside - Molecular Techniques: Tools for Success


    Hybridization - ELISA

    • Sensitive and specific

    • Quantification of
    picogram levels of protein

    de la Rica and Stevens, 2012

    From Bench to Bedside - Molecular Techniques: Tools for Success


    Array-based assays

    From Bench to Bedside - Molecular Techniques: Tools for Success


    Array-based Expression Profiling
    Hybridization targets are or
    microprinted on to glass slides chip-based arrays

    Blackburn and Shoko, 2011.

    From Bench to Bedside - Molecular Techniques: Tools for Success


    Array-based Expression Profiling

    Robinson et al., 2000


    From Bench to Bedside - Molecular Techniques: Tools for Success
    Protein Arrays
    Analytical: Antibody-based arrays for
    protein detection

    Functional: Protein-based arrays for


    detection of functional interactions

    Reverse Phase: Analyze many samples at


    once.

    Sutandy et al. 2013


    From Bench to Bedside - Molecular Techniques: Tools for Success
    From Bench to Bedside - Molecular Techniques: Tools for Success
    References

    • Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New
    York: Garland Science; 2002.
    • Armstrong, J. A. and Schulz, J. R. 2008. Agarose Gel Electrophoresis. Current
    Protocols Essential Laboratory Techniques. 00:7.2:7.2.1–7.2.20.
    • Gallagher, S., Winston , S. E., Fuller, S. A. and Hurrell, J. G. 2008. Immunoblotting
    and Immunodetection. Current Protocols in Molecular Biology. 83:III:10.8:10.8.1–
    10.8.28.
    • de la Rica, R. and Stevens, M. M. 2012. Nature Nanotechnology 7, 821–824.
    • Robinson, B.W.S., Erle, D.J., Jones, D.A., Shapiro, S. Metzger, W.J., Albelda, S.M.,
    Parks, W.C., Boylang A. 2000. Recent advances in molecular biological techniques
    and their relevance to pulmonary research. Thorax 55:329-339
    • Blackburn, J.M. and Shoko, A. Protein Microarrays: Methods and Protocols.
    Methods in Molecular Biology. 785:305-330.
    • Sutandy, F. R., Qian, J., Chen, C.-S. and Zhu, H. 2013. Overview of Protein
    Microarrays. Current Protocols in Protein Science. 72:27.1:27.1.1–27.1.16.

    From Bench to Bedside - Molecular Techniques: Tools for Success

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