Cloning and Sequencing of Danio rerio (zebrafish) Polycomb Gene Using Polymerase Chain Reaction (PCR) - PowerPoint PPT Presentation

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Cloning and Sequencing of Danio rerio (zebrafish) Polycomb Gene Using Polymerase Chain Reaction (PCR)

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Cloning and Sequencing of Danio rerio (zebrafish) Polycomb Gene Using Polymerase ... comparing gene and protein sequences against others in public databases (GenBank) ... – PowerPoint PPT presentation

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Title: Cloning and Sequencing of Danio rerio (zebrafish) Polycomb Gene Using Polymerase Chain Reaction (PCR)


1
Cloning and Sequencing of Danio rerio (zebrafish)
Polycomb Gene Using Polymerase Chain Reaction
(PCR)
  • Y-W Peter Lin, G. Packert, and Teresa R Petrino
  • School of Natural and Health Sciences, Barry
    University, Miami Shores, Florida

2
Polycomb gene (Pc)
  • Regulates gene expression by repressing other
    regulatory genes
  • Mechanism of gene repression is unknown
  • Important in embryo development
  • Mutation in Pc related to some human cancers
    and limb abnormalities

3
Why Zebrafish?
  • Easily bred and maintained

4
Extra-uterine development
Long term goal To evaluate the role of Polycomb
gene in regulating embryo development in
zebrafish
5
To Isolate Zebrafish Polycomb Gene
Needle in a Hay Stack
6
Degenerate Oligonucleotide-Primed Polymerase
Chain Reaction (DOP-PCR)
7
PCR Primer Design  
Degenerate Primers designed based on Regions of
Polycomb Pc2-like Proteins and Genes (in Human,
Mouse, Chicken and Frog) that show Conserved
Amino Acid and Conserved Nucleotide sequences
8
Polycomb-Specific Primer Design
Forward and Reverse Primers Magnets for fishing
out the needle from the hay stack
9
( K T/G  R G/A  N A/T/G/C )       ( M
A/C  Y C/T ) Degenerate Primers          
Polycomb F2 (Forward Primer)       5'      ATG
GAG CTG CCN GCN GT      3'                        
Target  (Amino Acids)                     M  
E   L   P   A   V                  Zebra R6
(Reverse Primer)       3'       TG GTR GTC ATG
GTC GGN K     5'                       Sense
Strand               5'       AC CAY CAG TAC
CAG CCN M  3'                      
Target  (Amino Acids)        H       
H        Q   Y   Q   P   
M methionine E glutamic acid L leucine P
proline A alanine V valine H histidine Q
glutamine Y tyrosine
10
PCR using Polycomb-specific Primers
Expected PCR product 400 bp
11
PCR Products
12
DNA Sequencing
University of Miami, DNA Core
13
BLAST Sequence Analysis
  • Basic Local Alignment Search Tool
  • National Center for Biotechnology Information
    (NCBI)
  • For comparing gene and protein sequences against
    others in public databases (GenBank)
  • Sequence homology (similarity) search

14
BLAST Search Results
  • Database
  • nr 661,919 sequences 208,475,209 total letters
  • Significant Homology to Polycomb (Pc) Protein

15
Conclusions
  • PCR products (403 bp) isolated, cloned and
    sequenced
  • Encoded predicted 134 amino acids of the
    Chromodomain of the Polycomb (Pc)
  • Zebrafish Pc 85 homology to Pc of Human, Mouse,
    Chicken and Frog

16
Submissions to GenBankZebrafish Polycomb-like
Gene (partial) Sequence Clone B1-Sp6
(Sense)submitted to GenBank Database (via
BankIt) on April 6, 2001.ACCESSION
BankIt395137 Nucleotide AY029300
(GI13625549),  Protein AAK31300 (GI13625550) DE
FINITION   Danio rerio Polycomb mRNA partial
cds.AUTHORS   Petrino,T.R., Lin, Y.-W.P.,
Packert,G.R., Nadal,C.N., Torrealday,S., and
Greene,D.A.
17
Acknowledgements
  • Research supported by NIH-MBRS SCORE Grant
    GM45455-08
  • NIH-MARC USTAR Grant GM08021-17
  • Barry University Scholarship Grant
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