Biochemistry SSheng Zhao ( 赵晟 ), Biochemistry and Molecular Department of Medical school in Southeast University WWeb: E or QQQ /MSN/Skype/gChat: MMobile: or Conception, theory, research, and application ——Logic and LIY (Learn It Yourself)

Chapter 2: Structure and Function of Nucleic Acid Property and technique of nucleic acid

Structural Changes in DNA Melting

Hybridization of Nucleic Acid

Denaturation & Renaturation of DNA pH, temperature or ionic strength can cause DNA to melt Absorbance at 260 nm increases as the bases unstack is called hyperchromic shift; T mThe midpoint temperature of the absorbance curve: melting temperature, T m. T mT m =81.5 o C+16.6 (lg[Na+])+0.41(fraction G+C)-0.63(% formamide)-(600/length) –More ionic strength, higher T m –More G/C, higher T m –Less formamide, higher T m –Longer, higher T m

Hydrolysis of Nucleic Acids RNA is resistant to dilute acid DNA is depurinated by dilute acid DNA is not susceptible to base RNA is hydrolyzed by dilute base

 hydrolysis  hydrolysis

Restriction Enzymes Bacteria have learned to "restrict" the possibility of attack from foreign DNA by means of "restriction enzymes" Type II and III restriction enzymes cleave DNA chains at selected sites Enzymes may recognize 4, 6 or more bases in selecting sites for cleavage An enzyme that recognizes a 6-base sequence is a "six-cutter"

Type II Restriction Enzymes No ATP requirement Recognition sites in dsDNA usually have a 2-fold axis of symmetry Cleavage can leave staggered or "sticky" ends or can produce "blunt” ends

Solid Phase Oligonucleotide Synthesis

DNA Replication  DNA is a double-helical molecule  Each strand of the helix must be copied in complementary fashion by DNA polymerase  Each strand is a template for copying  DNA polymerase requires template and primer  Primer: an oligonucleotide that pairs with the end of the template molecule to form dsDNA  DNA polymerases add nucleotides in 5'-3' direction

Polymerase chain reaction

Developed in 1983 by Kary Mullis, In 1993, Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith (developing site-directed mutagenesis) for his work on PCR.

Nucleic acid sequencing  First generation: single sequence, classic, accurate, stable, low throughput  Secondary generation: high throughput, short reads, lower accuracy  Third generation: single molecular sequencing, fast, high throughput, super long and high accurate reads

Chain Termination Method Based on DNA polymerase reaction  Run four separate reactions  Each reaction mixture contains dATP, dGTP, dCTP and dTTP, one of which is P- 32 -labelled  Each reaction also contains a small amount of one dideoxynucleotide: either ddATP, ddGTP, ddCTP or ddTTP

 First generation  Secondary generation  Third generation (BIG DATA)