Title: Cloning and Sequencing of Danio rerio (zebrafish) Polycomb Gene Using Polymerase Chain Reaction (PCR)
1Cloning 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
2Polycomb 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
3Why Zebrafish?
- Easily bred and maintained
4Extra-uterine development
Long term goal To evaluate the role of Polycomb
gene in regulating embryo development in
zebrafish
5To Isolate Zebrafish Polycomb Gene
Needle in a Hay Stack
6Degenerate Oligonucleotide-Primed Polymerase
Chain Reaction (DOP-PCR)
7PCR 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
8Polycomb-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
10PCR using Polycomb-specific Primers
Expected PCR product 400 bp
11PCR Products
12DNA Sequencing
University of Miami, DNA Core
13BLAST 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
14BLAST Search Results
- Database
- nr 661,919 sequences 208,475,209 total letters
- Significant Homology to Polycomb (Pc) Protein
15Conclusions
- 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
16Submissions 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.
17Acknowledgements
- Research supported by NIH-MBRS SCORE Grant
GM45455-08 - NIH-MARC USTAR Grant GM08021-17
- Barry University Scholarship Grant