Isolation of promoter and other regulatory elements

1. Isolation of promoter and other regulatory elements
COURSE MBB 601
SUBMITTED TO
Dr Shubha Banerjee
Assist. Professor
PMBB (IGKV)
SUBMITTED BY
Shailendra Pandey
PMBB (PhD Student)

2. Analogical

3. https://status.libretexts.org
The "Central Dogma"
In September 1957, Francis Crick gave a lecture in which he
outlined key ideas about gene function, in particular what he
called the central dogma.

5. Regulatory elements are short DNA sequence motifs that are recognized and bound by regulatory
proteins, namely transcriptional activators, repressors, terminators and initiators of replication.
From a position bound to their specific target sequence, regulatory proteins affect the general
transcription factors of the pre-initiation complex RNA polymerase II and its cofactors.
Regulatory elements can often be detected by mere sequence analysis, but confirmation of
binding of specific factors requires experimental evidence such as gelshifts (bandshifts).
Regulatory sequence (elements)
Typical regulatory elements can be defined as promoter, enhancer, silencer, insulator or boundary
element, matrix attachment region (MAR) or scaffold attach- ment region (SAR) and locus control
region (LCR)
The International Journal of Biochemistry & Cell Biology 35 (2003) 95–103

6. From Wikimedia Commons
Shafee T, Lowe R (2017). "Eukaryotic and prokaryotic gene structure". WikiJournal of Medicine 4 (1).
Gene structure eukaryote

8. Regulatory sequence (elements)
• CAAT box GGCCAATCT
• Pribnow box TATAAT
• TATA box TATA(A/T)A(A/T)
• A-box TGACTCT
• B-box TGTCTCA
• Z-box ATACGGT
• C-box GACGTC
• E-box GGCACGAGGC
• G-box CACGTG
https://en.wikipedia.org/wiki/Regulatory_sequence

9. Cis-regulatory element
Cis-regulatory elements (CREs) or Cis-regulatory modules
(CRMs) are regions of non-coding DNA which regulate the
transcription of neighboring genes.
Trans-acting
trans-acting (trans-regulatory, trans-regulation), in general,
means "acting from a different molecule"
In the context of transcription regulation, a trans-acting factor
is usually a regulatory protein that binds to DNA.

10. Isolation of promoter and regulatory elements
work flow
1. Identification
2. Isolation
3. validation

12. http://rsat.sb-roscoff.fr/

13. Overview of the main applications of RSAT, with associated input data types

15. http://rsat.france-bioinformatique.fr/teaching/tutorials.php

17. Methods for isolation of promoter and regulatory elements
• PCR based
• nested PCR
• Inverse PCR
• TAIL-PCR
• Whole Genome PCR
• Genome Walking
• Plasmid Rescue
• Promoter-probe libraries
• Artificially designed promoters
• FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements).

18. nested
PCR

19. M Basyuni et al., 2018
obtain promoter region of a triterpene synthase KcMS gene
the genomic DNA library was digested with
DraI (library 1), EcoRV (library 2), PvuIII
(library 3), and StuI (library 4) in separates tubes
to make a blunt end.
Walker adaptors were ligated to the digests, and the ligated
products were used as a template to amplify promoter regions of
KcMS gene.
The Universal Genome Walker kit (Clontech, USA) was used two specific
oligonucleotide primers: Kc-A1
(5'GATTTCCTCACCATCCTGAACCTTCC-3')
and Kc-A2
(5'-TCAAACTCCCATGTTTGCCTTCCCAGG3'),
were synthesized.
These primers were used alongwith two outer
adapter primers provided with the kit,
AP1
(5'-GTAATACGACTCACTATAGGGC-3')
and AP2 (5'-
CCCCATCCCTTCGAAGGTCGCAATCGC-3').
nested
PCR

20. he first PCR was performed using AP1 primer and Kc-A1 primer with following PCR conditions: 7 cycles
The nested PCR with AP2 and Kc-A2 primer was carried out with the first PCR product as template.
The PCR product of promoter fragment was separated using 1 % agarose gel
Fragment was ligated into a plasmid vector of TOPO TA cloning vector and propagated in Escherichia
coli,
nested
PCR

21. nested
PCR

22. nested
PCR

23. Inverse PCR (IPCR) for Obtaining Promoter Sequence
From:
Methods in Molecular Biology, vol. 130, Transcription Factor Protocols
Edited by: M. J. Tymms © 2000 Humana Press Inc., Totowa, NJ
Used to amplify DNA with only one known sequence.
PCR to be carried out even if only one sequence is available from
which primers may be designed.
Inverse PCR is especially useful for the determination of insert
locations. For example, various retroviruses and transposons
randomly integrate into genomic DNA.
The inverse PCR method involves a series of restriction digests and
ligation, resulting in a looped fragment that can be primed for PCR
from a single section of known sequence.

24. Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR)
The key feature of the TAIL-PCR strategy is the use of
two primer-sets that
differ in length and have different melting
temperatures (thermal asymmetry)
Insertion-specific primers are used together
with arbitrary degenerate primers (AD primers),
Designed to differ in their annealing temperatures.
Alternating cycles of high and low annealing
temperature yield specific products bordered by
an insertion-specific primer on one side and an
AD primer on the other.
specificity is obtained through subsequent
rounds of TAIL-PCR, using nested insertion-
specific primers

26. Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR)
TAIL cycling consists of 15 super cycles, in which each super
cycle consists of two high stringency cycles and one low
stringency cycle
During the high stringency cycles, the insertion-specific primer
binds preferably to its target sequences, and the resulting
product is linearly amplified (thermal asymmetry).
During the reduced stringency cycle, the lower anneal-
ing temperature allows also the AD primers to bind (thermal
symmetry),
high stringency products are converted into double-stranded
form. Thus, the number of specific target molecules for the next
round increases logarithmically.
In order to increase the yield of the specific type I product and
to decrease the amount of contaminating nonspecific products, a
second round of TAIL- PCR is performed using a nested
insertion-specific primer

27. Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR)

33. Formaldehyde is added directly to cultured cells. The
crosslinked chromatin is then
sheared by sonication and phenol-chloroform extracted.
Crosslinking be- tween histones and DNA (or between one
histone and another) is likely to dominate the chromatin
crosslinking profile
Covalently linked protein–DNA complexes are
sequestered to the organic phase, leaving only protein-
free DNA fragments in the aqueous phase.
For the hybrid- ization reference, the same procedure is performed
on a portion of the cells that had not been fixed with formaldehyde,
a procedure identical to a traditional phenol-chloroform extraction.
DNA resulting from each pro- cedure is then labeled with a
fluorescent dye, mixed, and comparatively hybridized to DNA
microarrays.
FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements)
To perform FAIRE, chromatin is crosslinked with
formaldehyde in vivo, sheared by sonication, and
phenol-chloroform extracted. The DNA recovered in
the aqueous phase is fluorescently labeled and
hybridized to a DNA microarray.

34. Electrophoretic Mobility Shift Assays
Technique used in the study of DNA-binding proteins
The principle of the assay is—DNA fragments and proteins
are mixed in a suitable buffer and binding is allowed to occur.
The mixture is then separated by nondenaturing gel
electrophoresis;
The mixture is then separated by nondenaturing gel
electrophoresis; stable complexes of DNA and protein are
generally significantly retarded in mobility in comparison with
the free DNA and the separated
complexes are normally viewed by phosphorimaging or
autoradiography
From: Methods in Molecular Biology, vol. 130, Transcription Factor Protocols
Edited by: M. J. Tymms © 2000 Humana Press Inc., Totowa, NJ

37. Q1. If both the repressor and activator are present and functional,
a. activate b. reppress
QN. When to stop asking why question in Molecular biology
a. once b. infinite c. never ask d. explain