汪世华分子生物学2

UV absorption
*Nucleic acids absorb UV light due to
the conjugated aromatic nature of the bases;
*The wavelength of maximum
absorption of light by both DNA and RNA is 260 nm (max= 260 nm), which is conveniently distinct from the max of protein(280 nm).
• Northern blotting involves the use of
electrophoresis to separate RNA samples by size and detection with a hybridization probe complementary to part of or the entire target sequence.
Chapter 2 DNA and Chromosome
2.1 DNA structure
Primary structure
(Nucleotide residue sequence)
Secondary structure
(DNA double helix)
Advance structure
(DNA supercoiling)
molecular biology for detection of a specific DNA sequence in DNA samples.
Southern blotting combines transfer
of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.
Notes
A telomere consists of up to hundreds of copies of a short repeated sequence (5’-TTAGGG-3’ in humans), which is synthesized by the enzyme telomerase.
Hybridization
Forming a double-stranded structure from two polynucleotide strands (either DNA or RNA) from different sources.
Southern blotting
A Southern blot is a method usedห้องสมุดไป่ตู้in
The telomeric DNA forms a special secondary structure, the function of which is to protect the
2.3 GENE
2.3.1 Classical concept
gene was mapped on
chromosomes.
activation of at least one gene requires that a regulatory sequence switch to the Z-DNA form.
The stability of a nucleic acid
helix stacked base pairs
The midpoint of the temperature range over which the strands of DNA separate is called the melting temperature (Tm). Marmur-Doty empirical formula:
Tm=69.3+0.41 (G+C) % Or
Northern blotting
• The northern blot is a technique used
in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample.
2.1.1 Primary structure
the nucleotide residue sequence of
the polynucleotide chain；
3’,5’-phosphodiester bond; write as 5’ →3’；(replication)
2.1.2 Secondary
DsNtrAucdtouurbele helix
A Variety of DNA
Structure
Notes
DNA can be induced to form an alternative helix, known as the A-form, under conditions of low humidity.
Linking number
*The linking number is the number of times
the two strands of a closed DNA duplex cross over each other. The linking number is always an integer, but may be positive or negative depending on the orientation of the two curves.
histone, H2A, H2B, H3, H4, and a further family, H1, which has some different properties, and a distinct role.
2.2.3 Telomere
A telomere is a region of repetitive nucleotide sequences at the end of a chromosome, which protects the end of the chromosome from deterioration or from fusion with neighboring chromosomes.
△ Lk = △ Tw + △ Wr
Twisting number &
*ThWe trwitihsteing number of a DNA is
the number of base pairs divided by the number of base pairs per turn of the double helix.
2.2.2 Histone
*The major protein components of
chromatin are the histones; small, basic (positively charged) proteins which bind tightly to DNA.
*There are four families of core
Tm=4(G+C)＋2(A+T)
Renaturation
The ability of the two separated complementary strands to reform into a double helix is called renaturation （Annealing）.
Notes
*Quantization of nucleic acids: 1 mg ml–
1 dsDNA has an A260 of 20. The corresponding value for RNA or ssDNA is approximately 25.
*Purity of DNA: The A260/A280 ratio of a
double-stranded DNA sample can be used to assess its purity. For pure DNA, the value is 1.8. Values above 1.8 suggest RNA contamination and those
Tm
2.2.1 Nucleosomes
The nucleosome core is the basic unit of chromosome structure, consisting of a protein octamer containing two each of the core histones, with 146 bp of DNA wrapped 1.8 times in a left-handed fashion around it.
H-bonds
Charge interactions
Denaturation and
Renature
The process of strand separation is called denaturation or melting.
Hyperchromicity : The UV absorption is increased when the two single DNA strands are being separated.
*The writhing number is the
number of times a duplex axis crosses over itself in space.
• Most natural DNA is negatively supercoiled, that is the DNA is deformed in the direction of unwinding
passing DNA through the break, and
Topoisomeras Break
LK Energy
es
Type I
one strand ± 1 -
2.2 Chromosome
Eukaryotic chromosomes each contain a long linear DNA molecule, which must be packaged into the nucleus. The name chromatin is given to the highly ordered DNA–protein complex which makes up the
2.1.3 Advance
•DNA supercoiling isstructure the coiling of the DNA axis upon itself, caused by a change in the linking number from Lk°, the value for a relaxed closed
the major importance of the A-form is that it is the helix formed by RNA, and by DNA-RNA hybrids.
Notes
Z-DNA is stable in synthetic double stranded DNA consisting purely of alternating pyrimidine-purine sequence.
Topoisomerases
*A DNA topoisomerase is an enzyme
that changes the number of times the two strands in a closed DNA molecule cross each other.
*It does this by cutting the DNA,