Transcript Lecture 8 LC710- 1st + 2nd hr

Hybridization of Nucleic Acids
DNA1
DNA2
RNA
Probe
Northern
hybridization
Southern
hybridization
Juang RH (2004) BCbasics
Preparation of Traditional Nucleic Acid Probe
Amino acid sequence
GLY-ASP-GLU-SER-SER-VAL-LEU----GGG-GAC-GAG-TCC-TCC-GTT-CTC---
*
*
*
*
Nucleic acid sequence
The nucleic acid sequence is
Deduced from amino acid sequence
Chemical synthesis
*
*
*
* Codon degeneracy
Synthesizing
oligonucleotide
PROBE: GGGGACGAGTCCTCCGTTCT
Juang RH (2004) BCbasics
Probe is labeled with radioactive 32P
32
P
GG
GGACG
AG
Hybridization
GG
T
TC
CT
CC
GTTCT CA
TGCTC
C
C
C
G
AGG
G
TC
A
A
G
AGGCAA
C
C
T
A
DNA
denaturation
Target gene
Single colony
Lysed
Juang RH (2004) BCbasics
Colony Is Screened by Hybridization with Probe
Colony hybridization
Transferring …
Collect filter
paper
Dissolve cell
Autoradiography
DNA denatured
Add probe
Juang RH (2004) BCbasics
Cover with
filter paper
Biochip Based on Hybridization
Sample DNA
Complementary DNA hybridize
Biochip
Each spot contains known DNA
Signal appears
Schena (2000) Microarray Biochip Technology, p. A31
Juang RH (2004) BCbasics
The Genetic Code
Initiation and termination Codons
– Initiation codon: AUG
– Termination codons: UAA, UAG, UGA
Degeneracy: partial and complete
Ordered
Nearly Universal (exceptions:
mitochondria and some protozoa)
Key Points
 Each of the 20 amino acids in proteins is specified by
one or more nucleotide triplets in mRNA. (20 amino
acids refers to what is attached to the tRNAs!)
 Of the 64 possible triplets, given the four bases in
mRNA, 61 specify amino acids and 3 signal chain
termination. (have no tRNAs!)
Key Points
 The code is nonoverlapping, with each nucleotide
part of a single codon, degenerate, with most amino
acids specified by two to four codons, and ordered,
with similar amino acids specified by related codons.
 The genetic code is nearly universal; with minor
exceptions, the 64 triplets have the same meaning in
all organisms. (this is funny)
Do all cells/animals make the same
Repertoire of tRNAs?
The Genetic Code
The Wobble Hypothesis:
Base-Pairing Involving the Third
Base of the Codon is Less Stringent.
Base-Pairing with Inosine at
the Wobble Position
In molecular biology, a wobble base pair is a non-WatsonCrick base pairing
between two nucleotides in RNA molecules.
The four main wobble base pairs are
guanine-uracil, inosine-uracil, inosine-adenine, and inosinecytosine (G-U, I-U, I-A and I-C).
The thermodynamic stability of a wobble
base pair is comparable to that of a
Watson-Crick base pair.
Wobble base pairs are fundamental in RNA
secondary structure and
are critical for the proper translation of the
genetic code.
Suppressor Mutations
Some mutations in tRNA genes alter the
anticodons and therefore the codons
recognized by the mutant tRNAs.
These mutations were initially detected as
suppressor mutations that suppressed the
effects of other mutations.
Example: tRNA mutations that suppress
amber mutations (UAG chain-termination
mutations) in the coding sequence of genes.
Making a (UAG) Mutation
Translation of an amber (UAG)
Mutation in the Absence of a
Suppressor tRNA
Translation of an amber Mutation in
the Presence of a Suppressor tRNA
Note it is amber su3…why?????????
Translation of an amber Mutation in
the Presence of a Suppressor tRNA
If there was a single tRNATyr gene, then could one
have a amber supressor of it?
Fig1
Oligonucleotide synthesis is carried out by a stepwise addition of nucleotide residues t o
the 5'-terminus of the growing chain until the desired sequence is assembled. Each
addition is referred to as a synthetic cycle (Scheme 6) and consists of four chemical
reactions:
* Step 1 - De-blocking (detritylation): The DMT group is removed with a solution of
an acid, such as T CA or Dichloroacetic acid (DCA), in an inert solvent (dichloromethane
or t oluene) and washed out , resulting in a free 5' hydroxyl group on the first base.
* Step 2 - Coupling: A nucleoside phosphoramidite (or a mixt ure of several
phosphoramidites) is activated by an acidic azole catalyst, tetrazole, 2-ethylthiotetrazole,
2-bezylthiotetrazole, 4,5-dicyanoimidazole, or a number of similar compounds. This
mixture is brought in contact with the starting solid support (first coupling) or
oligonucleotide precursor (following couplings) whose 5'-hydroxy group reacts with the
activated phosphoramidite moiety of the incoming nucleoside phosphoramidite to form a
phosphite triester linkage. This reaction is very rapid and requires, on small scale, about
20 s for its completion. The phosphoramidite coupling is also highly sensitive to the
presence of water and is commonly carried out in anhydrous acet onitrile. Unbound
reagents and by-products are removed by washing.
* Step 3 - Capping: After the completion of the coupling reaction, a small percentage
of the solid support-bound 5'-OH groups (0.1 t o 1%) remains unreacted and needs to be
permanently blocked from further chain elongation to prevent the formation of
oligonucleotides with an internal base deletion commonly referred to as (n-1) shortmers.
This is done by acetylation of the unreacted 5'-hydroxy groups using a mixture of acetic
anhydride and 1-methylimidazole as a catalyst. Excessreagents are removed by washing.
* Step 4 - Oxidation: The newly formed tricoordinated phosphite triester linkage is not
natural and is of limited stability under the conditions of oligonucleotide synthesis. The
treatment of the support-bound material with iodine and water in the presence of a weak
base (pyridine, lutidine, or collidine) oxidizes the phosphite triester into a
tetracoordinated phosphate triester, a protected precursor of the naturally occurring
phosphate diester internucleosidic linkage. This step can be substituted with a
sulfurization step to obtain oligonucleotide phosphorothioates (see below). In the lat ter
case, the sulfurization step is carried out prior to capping.
New Base
Are the proteins produced a
pure reflection of the mRNA
sequence????
tRNA environment, protein modifications post-translationally
Good things to Know
RNApol II
TATAA
CCATGG (Nco I site and Kozak Rule)
ATG
AGGT….splice
GT……………A………polypyrimidine AG
PolyA recog sequence
AATAAA
The Reasons why ATG is a single codon
and TGG is a single codon.
SELEX yields a functional binding site. A, COS7 cells were transfected in
triplicate with either pEBG or increasing concentrations of BENwt (250, 500,
and 1000 ng) and either p81TKluc (TK) luciferase reporter or p81TKluc-WT3X
(WT3X). The luciferase values are reported as relative luciferase activity
normalized to the amount of total protein. -Fold decrease in activity is measured
relative to the basal transcriptional activity observed with pEBG empty
expression vector alone. Western blot with anti-GST antibody shows dosedependent expression of GST-BEN.
B, COS7 cells were transfected in triplicate with either pEBG or
BENwt (1000 ng of each) and with either TK, or WT3X or Mut3X (600 ng of each)
and the Renilla construct (pRL-TK).