Studied by 62 peoplewhat is sanger/termination chain sequencing?
polymerization reaction that is interrupted
name 4 elements of sanger sequencing
# Labelled (radioactivity, luminescence…) forward primer (only)
# Polymerization with chain termination
# with dNTPs and ddATPs
=> where chain terminates tell you there is an A (becuase ddATP used)
PCR
size of PCR product - downstream application
RT-PCR
clone cDNA
quantitative PCR
quantify infection with DNA (cancer)
qRT-PCR
quantify infection with RNA virus (covid) quantify gene expression
what’s Dideoxynucleotide’s role in sanger sequencing
stops DNA polymerization: prevents formation of phosphodiester bond with next dNTP
how do you read sanger sequencing gel
5’-3’ from bottom to top
Automated sanger sequencing experiments
=> used if you want 1 sequence (~$5 for 1 sequence ~1,000bp)
=> verify the sequence of a plasmid
=> sequence one gene location
The general principle of next-generation sequencing
=> Used if you want millions of short sequences (~$8,000 for 1.5 billion sequences of ~150bpP
=> to sequence whole genomes
=> for experiments with whole genome coverage (such as RNAseq)
Long read sequencing
=> reads long DNA/RNA strands without amplification through an ion channel
# advantage = very long sequences, tiny machine
# limits = not very accurate
what is a H1 histone
also called linker histone - is not a core histone (= is not part of nucleosome) but binds DNA/nucleosome to promote condensation
what are structural proteins
(Structural Maintenance of Chromosomes, or SMC) are critical to form mitotic chromosomes
name 4 aspects of histones and nucleosomes
Histones are positively charged proteins
They form nucleosomes by assembling into an octamer (2x H2A, H2B, H3 and H4)
DNA wraps around nucleosomes (~200 pb per nucleosome, interaction between positive charges in histones and negative charges in DNA)
Histones have protruding tails
two things about histone tails
1) form contact with adjacent nucleosomes => compacts DNA to 30 nm filament => reduces accessibility to transcription factors.
2) can be modified
how do Chromatin remodeling complexes organize nucleosomes regulate gene expression
ejecting/adding nucleosomes (change nucleosome occupancy) => lower nucleosome occupancy = more accessible chromatin, and vice-versa.
reposition nucleosomes (slide a nucleosome along DNA) => enables to mask or expose a promoter
replace a canonical histone by a histone variant
what can H3 and H2a be replaced by
histone variants
what is a histone variant
a protein slightly different than the core histone, encoded by a different gene, that can replace the canonical histone in a nucleosome.
Examples of H3 variants are:
H3.3 (maintains open chromatin in actively transcribed regions)
CenPA (Stands for Centromeric protein A, loaded in chromatin at the centromere, enables kinetochore attachment)
Example of H2A variant:
H2AX (loaded into chromatin surrounding double strand breaks. Enable the recruitment of DNA repair proteins and thereby promotes DNA repair)
Histone tails (mostly N-ter tails) are
chemically modified
Modifications are
covalent and post-translational
Modifications can be
acetylation, methylation, phosphorylation and ubiquitination
Examples of writing convention for histone modifications:
H3K27ac (Acetylated Lysine #27 on histone H3)
H4K20me3 (Tri-methylated Lysine #20 on histone H4)
what are chemical modifications on histone tails called?
Marks
what do acetylation and methylation of lysine enable?
regulation of chromatin compaction/gene expression
what is acetylation generally associated with?
more open chromatin
what is methylation generally associated with?
closed chromatin.
what are histone tails modified by?
specific histone modifying enzymes.
some histone modifying enzymes ( ) the modification and some ( ).
add - remove.
histone code hypothesis
The theory that different histone modifications and their combination impacts gene expression.
histone marks are bound by ( ) and they
specific proteins (called “readers”) - they stabilize a close state.
(chromodomain proteins => ———) or ———
inhibit transcription - stabilize an open state.
(bromodomain proteins => ———-)
promote transcription
During Sanger sequencing (the original technique):
4 polymerization reactions are performed in parallel, each containing all 4 dNTPs plus one ddNTP
The primer used for polymerization is labelled.
The DNA segments obtained are separated in a regular agarose gel
During automated Sanger sequencing (the fluorescent one), the polymerization reaction contains
4 fluorescent ddNTPs and 4 non fluorescent dNTPs
A non-labelled primer
what sequencing application is Analyzing which bacteria are present in a field
nanopore sequencing
what sequencing application is RNA-seq after reverse transcription (quantification of all the RNA present in a sample)
next-generation sequencing
what sequencing application is Sequencing of a plasmid (one 600 bp DNA sequence needed)
Sanger sequencing
Each nucleosome contains:
A histone octamer composed of two copies of each of the following proteins: H2A, H2B, H3, and H4
if DNA compaction happens in the chromatin regions increases what happens to transcription in that region?
decrease
what place is H3.3 loaded into the chromatin?
in open chromatin
what place is H2A.X loaded into the chromatin?
around DNA double strand breaks
what place is CenP-A loaded into the chromatin?
at centromeres
chromatin remodelers
eject nuclesomes
add nuclesomes
move nuclesomes
replace histone with histone variants
steps of RNA seq
1: grow your cells in culture
2: extract mRNA
convert your mRNA into cDNA
Attach adaptors to your cDNA
Sequence by synthesis
compare the generated cDNA with the known genome
The amount of protein in a sample, or comparing relative amounts of protein in many samples
western blot
How much of a protein / modified protein (modified histone) is at a certain gene locus (certain part of a gene)
Chip qPCR
The global effect of something on transcription (expression)
RNA seq
How much of a protein / modified protein (modified histone) is all over the genome
Chip seq
Identifying splice variants, sequencing in a field, sequencing something over 10kb but less than 100 kb, you don’t need something super accurate, can directly sequence DNA or RNA, is portable.
Nanopore
Whole genome sequencing, used for RNAseq or chIP seq, can sequence DNA, or RNA after a reverse transcription. It is sequencing by synthesis.
illumina (next gen)
Sequencing something relatively small (less than 1,000 bp), verify the sequence of a plasmid, sequencing one gene location.
Automated sanger
Note
Flashcard