Biochem 4511 - Chapter 4
nucleic acids renature
- annealing is determined by base complementarity - the ability to re anneal is very important in nature and research
what stabilizes the DNA double helix
- base pairs form the core of the double helix and associate through H bonding - phosphate backbone form the periphery ( significant charge repulsion) - double helix formation is driven by base stacking (hydrophobic effect)
restriction enzymes in nature
- defend bacteria from infection by bacteriophage - bacteria methylate their own DNA - bacteriophages have unmethylated DNA - bacterial resection enzymes recognize and excise unmethylated viral DNA
Sanger DNA sequencing
- extension by DNA polymerase proceeds with a mixture of dNTPs and ddNTPs - once ddNTP is incorporated, extension stops - a mixture of products is generated -
nucleotide polymers
- individual nucleotides are connected through a phosphodiester bond
3 classes of RNA
- mRNA - tRNA - rRna
what drives DNA to form a double helix
- minimizes caged water (hydrophobic effect) - spreads out the negatively charged phosphate groups more - thermodynamically favored because it: →maximizes favorable interactions and bonds →separates charge →keeps polar group solvent exposed →keeping bases that can H bond solvent exposed →aromatic rings stacked on top of each other to prevent caged water
translation
- occurs in the ribosome, which contains rRNA and many other proteins - tRNA carries amino acids to the ribosome and bind to its complement in the mRNA template - amino acids are dictated by the Genetic Code
extension
- optimal temperature for heat stable DNA polymerases to work (varies based on which polymerase is used) - new strand is synthesized - 72ºC
annealing
- primers base pair to single stranded DNA - actual temperature depends on DNA template and primers - 55ºC
biological info encoded by a sequence of DNA is
- transcribed to RNA - translated into the amino acid sequence of a protein - central dogma
the genetic code
- universal - read mRNA sequence in genetic code by position of 3 nucleotides - redundancy : more than one code for many amino acids - specific tRNA for each three letter code - amount of each tRNA varies across species
DNA amplification by PCR
1. Denaturing 2. annealing 3. extension 4. repeat from step 1
nucleotides are comprised of three main components
1. base 2. sugar molecule 3. phosphate
PCR requires
1. heat stable polymerase (taq) 2. DNA template 3. DNA primers 4. temperature regulation
in a nucleotide the phosphate is attached to the
5' carbon in DNA and RNA - can attack to any hydroxyl group
bases are read in the
5' to 3' direction
sugar molecule
(backbone) deoxyribose (DNA) or ribose - pentose ( 5 C sugar)
DNA double helix
- 2 strands, wrapping around a common axis with right handed twist to form a double helix - the two strands run in opposite directions (antiparallel), but both are right handed - bases occupy the core of the helix - sugar- phosphate chains are on the outside - each base has a "Watson - Crick" pairing to generate a planar H bonded pair - DNA binding proteins get information from what is exposed in the major groove
DNA fragmentation
- DNA fragmented by enzymatic digestion can be analyzed to give sequence information - gel electrophoresis is a method of separating DNA fragments by size - DNA bands can be stained with chemicals such as ethidium bromide for fluorescent detection - ethidum bromide intercalates in between aromatic bases
transcription
- RNA polymerase unwinds and separates double stranded DNA at the position where transcription occurs - RNA product is complementary to the noncoding (template strand) - cells cannot thrive if there is no transcription
DNA sequencing
- a DNA molecule can be sequenced by using DNA polymerase to make a copy of a template strand - the sanger method for DNA sequencing involves the incorporation of dideoxynucleotides (ddNTPs) not a strand of DNA
purines
- adenine - guanine
Adenine pairs with
Thymine (T) - forms 2 hydrogen bonds
nucleotides
add phosphate group to the nucleoside
rRNA
aids in protein synthesis
recognition sequences
are palindromic in double stranded DNA, complementary on both sides
base
aromatic heterocyclic molecules that provide the name and identity for different nucleotides
bases definition
aromatic heterocyclic molecules which vary between nucleotides
phosphate
attached to the 5' carbon
nucleosides
base + pentose sugar
nucleotides
base + ribose + phosphate group that links nucleotides together (through the phosphate backbone) *- sugar + phosphate + base*
nucleosides
bases + a 5 membered sugar ring derives from ribose, these sugar groups provide the difference between DNA ( deoxyribonucleic acid) and RNA (ribonucleic acid) *-base + sugar* (no phosphate)
denaturation
double helix unfolds and dissociates at high temperatures
denaturing
double sanded DNA denatures and separates at high temperature to form single stranded DNA - 92-95ºC
mRNA
encodes protein sequences
restriction enzymes are
endonuclease that recognize a specific nucleotide sequence target in DNA and break the DNA chin at or near the target
congregated non polar molecules
fewer water molecules are ordered around the non polar molecules
restriction enzymes are used in
genetic engineering
deoxyribose
has an H on the 2' carbon
ribose
has an OH on the 2' carbon
DNA generated by PCR
is unmethylate which allows the use of restriction enzymes in processing of in vitro generated DNA - molecular cloning
translation
making proteins from an RNA template
separated non polar molecules
many caged water molecules are ordered around the non polar molecules
Tm
melting temperature
monomer unit of DNA and RNA
nucleotide
transcription occurs in the
nucleus
the hydrophobic effect
phenomenon by which non polar molecules aggregate to avoid contact with hydrophilic molecules, particularly water
translation of mRNA is completed by
the ribosome aided by tRNA
Dideoxynucleotides (ddNTPs)
they lack a 3' OH group - once incorporated into the replicating chain, no more NTPs can attach
in DNA fragmentation smaller pieces
travel the fastest and furthest towards the positive cathode
important nucleotides
Coenzyme A, ADP, and ATP are important metabolic molecules
guanine pairs with
Cytosine (C) - forms 3 hydrogen bonds
nucleic acids
DNA and RNA are polymers of nucleotides
restriction enzyme example
EcoR1 recognizes the double stranded sequence GAATTC and cleaves both stands at G↓AATTC - over hangs are called sticky ends
DNA amplification
PCR can be used to make billions of copies of DNA efficiently and accurately
aggregating non polar molecules releases caged water which is
VERY entropically favorable
tRNA
carries amino acids to the ribosome
restriction enzymes
class of proteins sho role is to cleave dsDNA often in a sequence specific manner - recognize a specific sequence in DNA and cut at a specific pattern
at lower temperatures
complementary polynucleotides anneal to native state - annexing is determined by base complementarity
transcription
converting DNA into RNA
reverse transcription
converting RNA to DNA
replication
copying DNA
pyrimidines
cytosine, thymine, uracil
bases are either
purine or pyrimidine
2 classes of bases
purines and pyrimidines (larger structure has smaller name)
DNA processing
restriction enzymes
genes
sequences of DNA