Biotechnology Project Lab

Transcription

1 Only for teaching purposes - not for reproduction or sale Advanced Cell Biology & Biotechnology Biotechnology Project Lab Giovanna Gambarotta

2 COMPETENCES THAT YOU WILL ACQUIRE - compare DNA sequences - identify exons and introns with BLAST or Ensemble - design primers for quantification or for cloning - design primers with additional restriction enzyme site to facilitate subcloning - plan a reverse transcriptase (RT) reaction with positive and negative controls - plan a polymerase chain reaction (PCR) with positive and negative controls - calculate the amount of the ingredients in a reaction, knowing the initial and the final concentration of each ingredient - project hybrid proteins fused with GFP - project proteins with a FLAG tag - analyze DNA sequences to identify possible mutations - plan subcloning from a vector to another vector - blunting sticky ends - use of annhyb to design primers and to organize and edit sequences - use of neb-cutter for restriction enzyme analysis and preparation of plasmid maps - use of Chromas to read an electropherogram - use of BLAST to compare sequences solve typical problems you can encounter in the laboratory life (dilution, quantification, ) Only for teaching purposes - not for reproduction or sale

3 October 23 rd 2017

4 December 18 th 2017

5 Genebank sequence analysis primer design RT-PCR cloning in the vector pcrii-blunt subcloning in different expression vectors sequence analysis Only for teaching purposes - not for reproduction or sale

6 Today aims: 1 - wellcome test 2 - exon-intron map 3 - comparison of sequences to identify specific domains 4 - how to use Annhyb to manage sequences

7 Only for teaching purposes - not for reproduction or sale NRG1 neuregulin1 (Mei and Xiong, 2008)

8 Only for teaching purposes - not for reproduction or sale paracrine/autocrine action juxtacrine action (Mei and Xiong, 2008)

9 Only for teaching purposes - not for reproduction or sale NRG1 receptors belong to the ErbB receptor family (Mei and Xiong, 2008)

10 Only for teaching purposes - not for reproduction or sale The ErbB signalling network (Yarden & Sliwkowski, 2001)

11 (Mei and Xiong, 2008) Only for teaching purposes - not for reproduction or sale

12 NRG1 alternative splicing isoforms Only for teaching purposes - not for reproduction or sale

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15 - which isoform would you choose to study juxtacrine interactions and why? Only for teaching purposes - not for reproduction or sale

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18 DQ (rat) Only for teaching purposes - not for reproduction or sale

19 Preliminary study of NRG1 sequences 1- download the three rat sequences of type III NRG1 from the ncbi site 2- copy the coding sequences and save them in annhyb, annotating where the ATG and STOP codons are 3-obtain the exon map of the first rat sequence (AF194439) and reply to the questions about exon map in the quiz 4-compare the three rat NRG1 type III sequences with BLAST to identify the sequences coding for alpha, beta, 1 and 3 exons. 5-load in moodle the sequence corresponding to alpha, beta, 1 and 3 exons Only for teaching purposes - not for reproduction or sale

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21 Preliminary study of NRG1 sequences 1- download the three rat sequences of type III NRG1 from the ncbi site 2- copy the coding sequences and save them in annhyb, annotating where the ATG and STOP codons are 3-obtain the exon map of the first rat sequence (AF194439) and reply to the questions about exon map in the quiz 4-compare the three rat NRG1 type III sequences with BLAST to identify the sequences coding for alpha, beta, 1 and 3 exons. 5-load in moodle the sequence corresponding to alpha, beta, 1 and 3 exons Only for teaching purposes - not for reproduction or sale

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24 coding sequence You can copy all the sequence, writing in the Description where ATG and STOP are, or you can copy only the coding sequence

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26 Only for teaching purposes - not for reproduction or sale

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29 Only for teaching purposes - not for reproduction or sale

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34 Only for teaching purposes - not for reproduction or sale

35 If you start from the A of ATG frame is 1.

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38 Preliminary study of NRG1 sequences 1- download the three rat sequences of type III NRG1 from the ncbi site 2- copy the coding sequences and save them in annhyb, annotating where the ATG and STOP codons are 3-obtain the exon map of the first rat sequence (AF194439) and reply to the questions about it in the quiz 4-compare the three rat NRG1 type III sequences with BLAST to identify the sequences coding for alpha, beta, 1 and 3 exons. 5-load in moodle the sequence corresponding to alpha, beta, 1 and 3 exons Exercise for the next lesson (Deadline November 5 th ): - comparing different rat sequences, please identify the EGF-like domain and the type I and type II domains and load them in moodle. Only for teaching purposes - not for reproduction or sale

39 If you have the cdna sequence, how can you identify the exons? mrna polya tail Only for teaching purposes - not for reproduction or sale

40 Genomic DNA Promoter exon1 Intron 1 exons 2,3, RNA polymerase RNA RNA splicing mrna polya tail Only for teaching purposes - not for reproduction or sale

41 Genomic DNA Promoter exon1 Intron 1 exons 2,3, RNA polymerase RNA RNA splicing mrna polya tail - if you align genomic DNA and cdna, the aligned sequences will correspond to exons - you can order the aligned sequences from the start query in order to have the exons from the first to the last Only for teaching purposes - not for reproduction or sale

42 Genomic DNA Promoter exon1 Intron 1 exons 2,3, mrna if you align genomic DNA and cdna, the aligned sequences will correspond to exons - you can order the aligned sequences from the start query in order to have the exons from the first to the last Only for teaching purposes - not for reproduction or sale

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47 If you go to Query start position, the exons will be in the right order from the ATG to the STOP

48 ????

49 Example: NRG1 type IIIβ3 Exon 1 Exon 2 Exon 3

50 Preliminary study of NRG1 sequences 1- download the three rat sequences of type III NRG1 from the ncbi site 2- copy the coding sequences and save them in annhyb, annotating where the ATG and STOP codons are 3-obtain the exon map of the first rat sequence (AF194439) and reply to the questions about it in the quiz 4-pairwise compare the three rat NRG1 type III sequences with BLAST to identify the sequences coding for α, β, 1 and 3 exons. 5-load in moodle the sequence corresponding to alpha, beta, 1 and 3 exons Only for teaching purposes - not for reproduction or sale

51 rat NRG1 sequences Only for teaching purposes - not for reproduction or sale

52 Only for teaching purposes - not for reproduction or sale

53 X

54 Sequence 1 ( Query ) Sequence 2 ( Sbjct ) Only for teaching purposes - not for reproduction or sale

55 Three rat NRG1 type III sequences When you compare AF with AF194438, the non aligned parts correspond to - the domain in the AF sequence - the domain in the AF sequence

56 Three rat NRG1 type III sequences When you compare AF with AF194438, the non aligned parts correspond to - the α domain in the AF sequence - the β1 domains in the AF sequence

57 Please, repeat the passages described in the slides, using the same sequences or other sequences to: identify the exon sequences coding for α, β, 1 and 3 domains submit them to moodle

58 Preliminary study of NRG1 sequences 1- download the three rat sequences of type III NRG1 from the ncbi site 2- copy the coding sequences and save them in annhyb, annotating where the ATG and STOP codons are 3-obtain the exon map of the first rat sequence (AF194439) and reply to the questions about it in the quiz 4-pairwise compare the three rat NRG1 type III sequences with BLAST to identify the sequences coding for alpha, beta, 1 and 3 exons. 5-load in moodle the sequence corresponding to α, β, 1 and 3 exons Only for teaching purposes - not for reproduction or sale

59 rat NRG1 sequences Only for teaching purposes - not for reproduction or sale

60 Remember: you can obtain an exon-intron map also from Ensembl, but the map is not available for all the genes

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63 Today s Student Learning Outcomes (SLOS) USE OF KNOWLEDGE AND LEARNING SKILLS - how to use Annhyb to save and manage sequences - how to use BLAST to compare sequences - how to get exon map of a cdna - how to identify specific domains within a cdna