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
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.






07 03 jpg
07_03.jpg gel electrophoresis.


  • 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.


07 05 jpg
07_05.jpg (RSPs) and varaible number tandem repeats (VNTRs)


07 05 2 jpg
07_05_2.jpg (RSPs) and varaible number tandem repeats (VNTRs)


07 06 jpg
07_06.jpg (RSPs) and varaible number tandem repeats (VNTRs)


07 07 jpg
07_07.jpg (RSPs) and varaible number tandem repeats (VNTRs)


07 08 jpg
07_08.jpg (RSPs) and varaible number tandem repeats (VNTRs)


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.


07 09 jpg
07_09.jpg libraries) and establishing their structure



07 10 jpg
07_10.jpg used to identify coding sequences:


07 10 2 jpg
07_10_2.jpg used to identify coding sequences:


07 11 jpg
07_11.jpg used to identify coding sequences:


07 11 2 jpg
07_11_2.jpg used to identify coding sequences:


-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.


07 12 jpg
07_12.jpg 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.


07 12 2 jpg
07_12_2.jpg 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.


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


07 13 jpg
07_13.jpg S1 nuclease protection or primer extension.


3. Studying gene expression S1 nuclease protection or primer extension.

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


07 14 jpg
07_14.jpg S1 nuclease protection or primer extension.


07 15 jpg
07_15.jpg S1 nuclease protection or primer extension.


07 16 jpg
07_16.jpg S1 nuclease protection or primer extension.


07 17 jpg
07_17.jpg S1 nuclease protection or primer extension.


07 18 jpg
07_18.jpg S1 nuclease protection or primer extension.


07 19 jpg
07_19.jpg S1 nuclease protection or primer extension.


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