DNA Sequencing of the Northern Star Coral (Astrangia poculata) from New Jersey Artificial Reefs.
Nayimisha Balmuri and Dr. Peter F. Straub
Richard Stockton College
School of Natural Sciences and Mathematics
ABSTRACT: The purpose of this study was to investigate the molecular evolution of the Northern Star coral, Astrangia poculata, on shipwrecks off of Atlantic City. Individual coral colonies were sampled from several collection sites
offshore of Atlantic City, NJ at depths from 20-30 m by a scuba diver. Between 10-30 mg of tissue was extracted from individual polyps using a DNA extraction kit (Qiagen Corp). DNA was amplified by the polymerase chain reaction
(PCR) using eukaryotic universal primers for the small subunit (18s) ribosomal RNA gene. PCR products were cloned in pGemT (Promega) and plasmid preparations were cycle sequenced to determine the DNA base sequence of the
individual sample.The DNA sequence was found to be a match for A.poculata in one sample out of the eight.
INTRODUCTION: Astrangia poculata, the Northern Star Coral (Fig 1), is
found from the sub-tropics to the temperate zone on the Northwest
Atlantic Coast (Fig 4). Our local coastal ocean is consists primarily of gently
sloping sandy plains, therefore, Astrangia poculata is found on ship wrecks.
The Individual coral colonies were sampled from collection sites offshore of
Atlantic City at depths from 20-30 m. The samples used in this experiment are
from PETWreck, Site10.
Figure 1: Beautiful Northern Star Coral in the Ocean
PURPOSE: The purpose of this study was to contribute DNA sequences to an
ongoing investigation of the distribution and molecular evolution of the Northern
Star coral (Astrangia poculata).
RESULTS: Cloning of the PCR product and DNA sequencing was successful.
BLAST search shows that the DNA for sample 3 is a match (320 bp) for the
Northern Star Coral 18s small ribosomal subunit. The E value was 3.1 x e^-139.
The alignment of the PCR sequence with Astrangia poculata (danae) is shown in
Figure 8.
FURTHER RESEARCH: Of the four samples, one PCR sequence was shown to
be cloned and sequenced. In the future, the other samples will need to be
recloned and sequenced from the clean PCR product obtained from this study.
These sequences will contribute the larger study of the molecular evolution and
distribution of A. poculata in the Atlantic Ocean.
Figure 3: Copral
heads before
extraction
Figure 2: Retrieval
of Northern Star
Coral from the
Atlantic Coast of NJ
METHODS: Individual coral colonies were sampled from several collection sites
offshore of Atlantic City, NJ (Fig 2). 10-30 mg of tissue was extracted and purified
from individual polyps using a DNeasy Blood and Tissue extraction kit. Genomic
DNA was quantified by UV spectrophotometer and gel analysis and amplified by
the polymerase chain reaction using eukaryotic universal primers for a portion of
the small subunit (18s) ribosomal RNA gene. PCR products were analyzed by
agarose gel electrophoresis (Fig 5) and then purified using Wizard SV Gel and
Promega PCR Clean-up kit (Fig. 6). Bio-Rad Experion DNA chip analysis was used
to evaluate the PCR product in each sample. Purified PCR products were then
ligated into the plasmids using a pGemT cloning kit. Ligation products were
transformed into ultra-competent JM109 E. coli cells by heat shock for 50 s at 42º
C, grown for 90 min in LB broth with shaking. Transformants were then plated on
LB agar medium supplemented with 100 µg/ml ampicillin and 80 µg/ml X-Gal
and 0.5 mM IPTG for blue-white color selection and grown overnight at 37º C.
Individual white colonies were picked and grown for five days at 37º C in 5 ml
broth cultures of LB medium with 50 µg/ml ampicillin. Plasmid DNA was
prepared using Wizard Plus Miniprep DNA Purification System (Promega) and
then quantified on a UV spectrophotometer. The plasmid DNA was also analyzed
by agarose gel electrophoresis to ensure presence of DNA ( Fig 7). A Genome Lab;
Dye Terminator Cycle Sequencing quick start kit (Beckman) was used with plasmid
preparations for each reaction plus T7 or SP6 flanking sequencing primers and was
subjected to PCR cycling reactions. After cycling, sequence reactions were purified
by ethanol precipitation, dried and resuspended in sequence loading solution. The
samples were sequenced on a Beckman Coulter CEQ 8000 automated capillary
genetic analyzer. Sequences were uploaded to the BLAST server at the National
Center for Biotechnology Information and compared with known sequences in
GenBank.
Figure 4: Locations of Northern Star Coral along NJ’s Atlantic
Coast
Figure 5: Gel Analysis of PCR
products
Figure 6: Clean PCR
product
Figure 7: Gel Analysis of
Plasmid DNA
Figure 8: Alignment of PCR sequence with A. poculata
(danae)
Special thank you to Dr. Peter Straub, Rachel Sura, Richard Stockton College, NAMS, NSF
(0619611) for their support in this study