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