Polymerase Chain Reaction and DNA Sequencing ?? · PPT file · Web view · 2016-05-03Polymerase Chain Reaction (PCR) ... PowerPoint Presentation PowerPoint Presentation PowerPoint Presentation RT-PCR PowerPoint Presentation PowerPoint Presentation PowerPoint Presentation PowerPoint Presentation ...

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  • Polymerase chain reaction:

    Starting with VERY SMALL AMOUNTS OF DNA (sometimes a few molecules), one can amplify the DNA enough to detect it by electrophoresis.

    Polymerase Chain Reaction (PCR)

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  • Rate of PCR 2n

    InitialDNA

    8

    4

    2

    1

    Number of DNA molecules

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  • Copies of DNA=2N

    Sheet1

    CYCLE NUMBERDNA copy number

    01

    12

    24

    38

    416

    532

    664

    7128

    8256

    9512

    101,024

    112,048

    124,096

    138,192

    1416,384

    1532,768

    1665,536

    17131,072

    18262,144

    19524,288

    201,048,576

    212,097,152

    224,194,304

    238,388,608

    2416,777,216

    2533,554,432

    2667,108,864

    27134,217,728

    28268,435,456

    29536,870,912

    301,073,741,824

    311,400,000,000

    321,500,000,000

    331,550,000,000

    341,580,000,000

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    PCR CYCLE NUMBER

    AMOUNT OF DNA

    Sheet2

    PCR CYCLE NUMBER

    AMOUNT OF DNA

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    Sheet3

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  • RT-PCR

    Polymerase chain reaction amplification of cDNA can also be used to detect specific transcripts in a RNA sample. In this procedure, known as RT-PCR, reverse transcriptase is used to copy all of the mRNAs in an RNA sample into cDNA. Usually, oligo dT molecules, that anneal to the poly A tails of the mRNA, are used as primers. This single stranded cDNA can then be amplified by PCR using primers that anneal to a specific transcript sequence.

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  • RT-PCR

    AAA(A)n

    5-Cap

    mRNA

    (dT)12~18

    primer

    anneal

    3

    5

    Reverse transcriptase

    dNTP

    5

    cDNA:mRNA hybrid

    Regular

    PCR

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  • The amplified DNA fragments that are produced can by analyzed by agarose gel electrophoresis.The amount of amplified fragment produced is proportional to the amount of target mRNA in the original RNA sample. Although less quantitative than Northern blots, RT-PCR is extremely sensitive and can be used to detect very rare mRNA species.

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  • How do we accurately quantify the amount of DNA?

    Real-time PCR

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  • Real Time PCR

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  • 2a. excitation filters

    2b. emission filters

    1. halogen tungsten lamp

    4. sample plate

    3. intensifier

    5. ccd detector 350,000 pixels

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  • Amplification Plot of real-time PCR

    DNA copy number (log)

    PCR cycle (Ct)

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  • DNA sequencing

    The Sanger di-deoxy method involves the synthesis of DNA by a DNA polymerase.DNA synthesis is terminated at specific nucleotides by the incorporation of di-deoxy nucleotides that are missing the 3 OH.

    Sequencing is used to determine the precise order of nucleotides in a DNA molecule.

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  • Sequence analysis

    Four different reactions produce DNA fragments that are terminated (randomly) at each of the four nucleotides.These samples are resolved by electrophoresis.The shortest fragments, those terminated closest to the primer, run faster than the longer fragments.

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  • A C G T

    DNA sequencing reactions can be radioactively labeled.Bands detected by X-ray film exposure.Sequence can be read in the 5 to 3 direction from the bottom of the image towards the top.

    G

    C

    A

    A

    T

    A

    C

    T

    A

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  • This is great but

    Wouldnt it be great to run everything in one lane?

    Saves space and time, more efficient

    Fluorescently label the ddNTPs so that they each have a different color

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  • 07_03.jpg

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    07_03.jpg

  • Automated DNA sequencers use dideoxy terminators labeled with four different fluorescent dyes.All four reactions can be carried out simultaneously in a single reaction.

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  • Fluorescently tagged fragments are resolved by capillary electrophoresis and detected by a flourometer. The DNA sequence is read automatically.

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  • Beckman CEQ 2000, 8 capillary

    ABI Prism 3730, 96 capillary

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  • Affymetrix Gene Chip

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  • Affymetrix Expression Arrays

    http://www.affymetrix.com/technology/ge_analysis/index.affx

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  • Analysis of microarrays

    Microarrays allow for the simultaneous analysis of the expression of thousands of mRNAs.Useful for determining changes in gene expression patterns from one sample tissue to another.For example, microarrays have been used to study differences in gene expression in different tumor tissues.

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  • Genes with similar expression patterns are clustered together.

    Gene expression patterns can be associated with different

    disease patterns.

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  • Microarray example #1: Biomarker identification - lung cancer

    Samples

    Genes

    Garber, Troyanskaya et al. Diversity of gene expression in adenocarcinoma of the lung. PNAS 2001, 98(24):13784-9.

    8.psd

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    Gene expression patterns segregate the four major morphological lung tumor subtypes

    Patterns of gene expression promise to refine traditional morphologic classification of lung cancer.

    Can we distinguish subsets of genes that would allow molecular-level classification of tumor subtypes?

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    07_03.jpg

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    Gene expression patterns segregate the four major morphological lung tumor subtypes

    Patterns of gene expression promise to refine traditional morphologic classification of lung cancer.

    Can we distinguish subsets of genes that would allow molecular-level classification of tumor subtypes?

    CYCLE NUMBERDNA copy number

    01

    12

    24

    38

    416

    532

    664

    7128

    8256

    9512

    101,024

    112,048

    124,096

    138,192

    1416,384

    1532,768

    1665,536

    17131,072

    18262,144

    19524,288

    201,048,576

    212,097,152

    224,194,304

    238,388,608

    2416,777,216

    2533,554,432

    2667,108,864

    27134,217,728

    28268,435,456

    29536,870,912

    301,073,741,824

    311,400,000,000

    321,500,000,000

    331,550,000,000

    341,580,000,000

    0.0E+00

    2.0E+08

    4.0E+08

    6.0E+08

    8.0E+08

    1.0E+09

    1.2E+09

    1.4E+09

    1.6E+09

    1.8E+09

    0

    5

    10

    15

    20

    25

    30

    35

    PCR cycle

    DNA copy number

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