Chapter 7 analyzing dna and gene structure variation and expression
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Chapter 7 Analyzing DNA and gene structure, variation and expression. Sequencing and genotyping DNA Standard/manual DNA sequencing using dideoxynucleotide chain terminators ddATP, ddGTP, ddCTP, and ddTTP. . 07_01.jpg. 07_02.jpg. 07_02_2.jpg.

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Chapter 7 Analyzing DNA and gene structure, variation and expression

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Chapter 7 analyzing dna and gene structure variation and expression

Chapter 7Analyzing DNA and gene structure, variation and expression

  • Sequencing and genotyping DNA

  • Standard/manual DNA sequencing using dideoxynucleotide chain terminators ddATP, ddGTP, ddCTP, and ddTTP.


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Chapter 7 analyzing dna and gene structure variation and expression

  • Automated DNA sequencing using fluorophores and capillary gel electrophoresis.


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Chapter 7 analyzing dna and gene structure variation and expression

  • Simple/basic genotyping by restriction site polymorphisms (RSPs) and varaible number tandem repeats (VNTRs)

    - RSPs: single nucleotide polymorphism may cause a loss or gain in a restriction site generating an RSP. Used in identifying carriers for some disease causing genes.

    - VNTR: use of PCR or Southern blot hybridization to identify differences in the number of microsatellite tandem repeats.


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Chapter 7 analyzing dna and gene structure variation and expression

2. Identifying coding sequences (genes) in cloned DNA (e.g. libraries) and establishing their structure

  • Three features distinguish coding DNA from non-coding DNA:

    -i- coding sequences are highly conserved

    -ii- presence, in coding sequences, of open reading frames (ORFs).

    -iii- vertebrate coding sequences are often associated with CpG islands.

  • Routine/traditional methods for identifying evolutionary conserved coding sequences include zooblots. Recently, homology searching of sequence databases became a useful tool.


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Chapter 7 analyzing dna and gene structure variation and expression

  • Besides routine methods, new more specialized procedures are used to identify coding sequences:

    -i- Exon trapping uses an artificial RNA splicing assay.

    -ii- cDNA selection by heteroduplex formation using magnetic beads capture identifies expressed sequences in genomic clones.


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Chapter 7 analyzing dna and gene structure variation and expression

-iii- To obtain full length cDNA of any gene, a cDNA library is screened using a probe (an oligonucleotide of the gene) and a set of overlapping truncated cDNA clones are produced. Then, RACE-PCR (rapid amplification of cDNA ends) is a technique used to extend the 5’ and/or 3’ ends of a short cDNA clone to onbtain a full length cDNA.


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-iv- Mapping transcription start site could be achieved by S1 nuclease protection or primer extension.


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Chapter 7 analyzing dna and gene structure variation and expression

3. Studying gene expression

  • Principles of expression screening – in vitro versus in vivo. RNA analysis versus tissues and individual cells.


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