Escolar Documentos
Profissional Documentos
Cultura Documentos
I. Gel electrophoresis
III. Sequencing
IV. Next Generation
Sequencing Technologies
GEL ELECTROPHORESIS
GEL ELECTROPHORESIS
Charged molecules are separated in
response to an electric field
Negative electrode
Shorter fragments
travel faster through the
gel.
–
GEL ELECTROPHORESIS
FACTORS AFFECTING MIGRATION
Electrical charge
Voltage applied
IV. Blotting
V. Probe labeling
VII. Detection
SOUTHERN BLOTTING
I. DNA extraction and restriction
digestion
to isolate the gene of interest
DENATURATION (NaOH)
dsDNA ssDNA
Sugar-phosphate backbone is
cleaved
SOUTHERN BLOTTING
IV. Blotting
Transfer methods:
Capillary
Electroblotting
Vacuum or positive pressure blotting
Probe labels:
Radioactive isotopes
Non-radioactive labels
SOUTHERN BLOTTING
V. Probe labeling
Radioactive isotopes:
DISADVANTAGES:
Short half life
Radioactive waste
Access to dark room
SOUTHERN BLOTTING
V. Probe labeling
Non-radioactive labels:
Biotin, digoxygenin,
enzymes, antibodies
SOUTHERN BLOTTING
VI. Hybridization and washing
Non-radioactive labels:
Colorimetric
Fluorescent
Chemiluminescent
SEQUENCING
SEQUENCING
atgttgtatttgtctgaagaaaataaat
Why do we sequence DNA/ genome? ccgtatccactccttgccctcctgataa
gattatctttgatgcagagaggggggag
tacatttgctctgaaactggagaagttt
To identify genes in the genome tagaagataaaattatagatcaagggcc
agagtggagggccttcacgccagaggag
aaagaaaagagaagcagagctaggctct
To understand evolution of genes and
genomes
ssDNA as template
Oligonucleotide primers
DNA polymerase
Dideoxynucleotide triphosphates
(ddNTPs)
Visualized by UV light
SEQUENCING
2. Sanger chain termination method
Development of automated
sequencer
Uses fluorescent tags, one
for each base
Adenine
Thymine
Guanine
Cytosine
Sequence is recorded as
chromatogram
SEQUENCING
NEXT GENERATION SEQUENCING (NGS)
TECHNOLOGIES
NGS TECHNOLOGIES
Measured by chemiluminescence
NGS TECHNOLOGIES
NGS TECHNOLOGIES
2. SEQUENCING BY SYNTHESIS (ILLUMINA-SOLEXA)
Hybridization to beads
Amplification by emPCR
1 %,
Ion Torrent 200-400 bp 50 Mb – 15 Gb 2 – 7 hrs 25-3,500
insertion/deletion
~12 % insertion/
Oxford Nanopore ~200 kb 1.5 Gb – 4 Tb Up to 48 hrs 750
deletion
Brown, TA. (2001). Southern Blotting and Related DNA Detection Techniques. eLS. DOI: 10.1038/npg.els.0000996
Dingman, CW, MP Fisher, and T Kakefuda. (1972). Role of molecular conformation in determining the electrophoretic properties of
polynucleotides in agarose-acrylamide gels. II. Biochem. 11, 1242 – 1250.
Goodwin, S, JD McPherson, WR McCombie. (2016). Coming of age: ten years of next-generation sequencing technologies. Nature Reviews:
Genetics 2016; 17:333-351
Liu, L, Y Li, S Li, N Hu, Y He, R Pong, D Lin, L Lu, M Law. (2012). Comparison of Next-Generation Sequencing Systems. Journal of Biomedicine and
Biotechnology Volume 2012, Article ID 251364
Magdeldin, S. (2012). Gel Electrophoresis – Principles and Basics. InTech, Rijeka, Croatia. ISBN 978-953-51-0458-2
Ravi, I, M Baunthiyal, J Saxena (eds). Advances in Biotechnology. Springer India 2014. ISBN 978-81-322-1554-7 (eBook) DOI 10.1007/978-81-
322-1554-7
Wall, WJ. (2002). Techniques for DNA Analysis. Ullmann's Encyclopedia of Industrial Chemistry. DOI: 10.1002/14356007.e26_e01