ACS Study Prep

Know the different DNA technology techniques (Chapter 9)

Gel blotting is a technique for visualizing a particular subset of
macromolecules proteins, or fragments of DNA or RNA initially present in a
complex mixture
The steps involved in gel blotting are,
1. Separating the molecules by electrophoresis. This is done in a gel
which allows the molecules to migrate under the influence of an
electric field
2. The resulting electrophoresis is then blotted with a nitrocellulose
filter. The molecules stick tightly to the filter and will retain their
relative positions when flooded with fluid at the next step
3. The filter is then bathed with a solution containing a probe
The probe will combine specifically with the target molecules,
that is, the ones that you are looking for
The probe carries a mean of visualization (either a radioactive or
fluorescent marker)
The procedure for detecting DNA fragments containing a particular sequence
is as follows
1. DNA is extracted from the cell
2. The DNA is partially digested by a restriction endonuclease also
called restriction enzymes, recognize and cleave DNA at specific
sequences (recognition sequences or restriction sites) to generate a
set of smaller fragments
3. The resulting DNA fragments are separated by electrophoresis and
then denatured to form single stranded molecules (ssDNA)
4. Without altering their positions, the separated bands of ssDNA are
transferred to a nitrocellulose filter and exposed to a radiolabeled
cDNA or RNA
5. If the probe detects complementary DNA sequences, it will bind to
them
The different types of blots are as follows:

Type of Blot

Molecules separated by
electrophoresis

Probe

Southern

ssDNA

cDNA or RNA

Northern

denatured RNA

RNA or cDNA

Western

Protein

Antibodies

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/G/GelBlotting.html
The northern blot is a technique used in molecular biology research to
study gene expression by detecting RNA (or isolated mRNA) in a sample
Gel electrophoresis is a technique used for the separation of DNA, RNA, or
protein molecules using an electric field applied to a gel matrix
The Polymerase chain reaction (PCR) involves amplifying specific DNA
sequences
o The process involves amplifying the number of copies of a particular
DNA segment

o
o

Its a technique in molecular biology to amplify a single or few copies

of a piece of DNA across several orders of magnitude, generating
thousands to millions of copies of a particular DNA sequence
The process involves and relies on thermal cycling, consisting of cycles
of repeated heating and cooling of the reaction for DNA melting and
enzymatic replication of the DNA. Primers (short DNA fragments)
containing sequences complementary to the target region along with a
DNA polymerase are key components to enable selective and repeated
amplification
As PCR progresses the DNA generated is itself used as a template for
replication, setting in motion a chain reaction in which the DNA
template is exponentially amplified
1. DNA strands are separated by heating
2. The DNA strands are annealed to an excess of short synthetic
DNA primers that flank the region to be amplified
3. New DNA is synthesized by polymerization

Know the genetics aspect of biochemistry

A triplet codon in a nucleic acid sequence usually specifies a single amino
acid
The genetic code defines a mapping between tri-nucleotide sequences, called
codons, and amino acids
DNA (deoxyribonucleic acid) is a nucleic acid that contains the genetic
instructions used in the development and functioning of all known living
organisms and some viruses
o The main role of DNA molecules is the long-term storage of information
o DNA consists of two long polymers of simple units called nucleotides,
with backbones made of sugars and phosphate groups joined by ester
bonds
o The two strands run in opposite directions to each other and are
therefore anti-parallel
o Attached to each sugar is one of four types of molecules called bases
o It is the sequence of these four bases along the backbone that encodes
information
o This information is read using the genetic code, with specifies the
sequence of the amino acids within proteins
RNA (ribonucleic acid) is a biologically important type of molecule that
consists of a long chain of nucleotide units
o Each nucleotide unit consists of a nitrogenous base, a ribose sugar,
and a phosphate
o RNA is transcribed from DNA by enzymes called RNA polymerases, and
is generally further processed by other enzymes
o RNA is central to protein synthesis
Differences between RNA and DNA
o RNA is usually single stranded, while DNA is usually double stranded
RNA a much shorter chain of nucleotides
o RNA nucleotides contain ribose, while DNA contains deoxyribose (a
type of ribose that lacks one oxygen atom)

These hydroxyl groups makes RNA less stable than DNA because
it is more prone to hydrolysis
o RNA has the base uracil, while DNA has the base thymine
The complementary base to adenine Is not thymine, as it is DNA,
but rather uracil, which is an unmethylated form of thymine
Ribosomal RNAs (rRNAs) are components of ribosomes the complexes that
carry out the synthesis of proteins
Messenger RNAs (mRNAs) are intermediaries, carrying out genetic
information from one or a few genes to a ribosome, where the corresponding
proteins can be synthesized
Transfer RNAs (tRNAs) are adapter molecules that faithfully translate the
information in mRNA into a specific sequence of amino acid

Know what Okazaki fragments are

Okazaki fragments are relatively short fragments of DNA (with no RNA
primer at the 5 terminus) created on the lagging strand during DNA
fragments
o When the lagging strand is being replicated on the original strand, the
5-3 pattern must be used; thus a small discontinuity occurs and an
Okazaki fragment forms
o These fragments are processed by the replication machinery to
produce a continuous strand of DNA and hence a complete daughter
DNA helix
o In dealing with the synthesis of complementary DNA strands, the
emerging leading strand always reads 3 to 5. The antiparallel
complement strand, the emerging lagging strand reads 5 to 3
Because the original strands of DNA are antiparallel, and only
one continuous new strand can be synthesized at the 3 end of
the leading strand due to the 5-3 polarity of DNA polymerases,
the other strand must grow discontinuously in the opposite
direction
Regarding the lagging strand, the result of this strands
discontinuous replication is the production of a series of short
sections of DNA called Okazaki fragments
Each Okazaki fragment is initiated near the replication fork at an
RNA primer created by a primase, and extended by DNA
polymerase III
Conservative versus semiconservative
DNA replication is semiconservative
o Each DNA strand serves as a template for the synthesis of a new
strand, producing two new DNA molecules, each with one new strand
and one old strand
o Semiconservative replication would produce two copies that each
contained one of the original strands and one new strand
o Conservative replication would leave the two original template DNA
strands together in a double helix and would produce a copy composed
of two new strands containing all of the new DNA base pairs

Know about enzymes and restrictive enzymes

Enzymes are proteins that catalyze (increase the rates of) chemical reactions
o In enzymatic reactions, the molecules at the beginning of the process
are called substrates, and the enzyme converts them into different
molecules, called the products
o Enzymes acts as an catalyst and work by lowering the activation
energy for a reaction, thus increasing the rate of the reaction
o Enzyme activity can be affected by other molecules
Inhibitors are molecules that decrease enzyme activity,
activators are molecules that increase enzyme activity
Drugs and poisons are enzyme inhibitors
Activity is affected by temperature, chemical environment, and
the concentration of substrate
o Enzymes are very specific as to which reactions they catalyze, and the
substrates that are involved in these reactions
Complementary size, charge, and hydrophilic/hydrophobic
characteristics of enzymes and substrates are responsible for
this specificity
Cofactors are components of enzymes that some enzymes require. These
non-protein molecules are bound to some enzymes for activity
Coenzymes are small organic molecules that transport chemical groups from
one enzyme to another
Restriction enzymes, or restriction endonuclease, is an enzyme that
cuts double-stranded or single DNA at specific recognition nucleotide
sequences known as restriction sites
Know about hybridization
DNA Hybridization is taking two DNA samples to be compared. They are
completely denatured by heating. Then when two solutions mixed and slowly
cooled, DNA strands of each sample associate with their normal
complementary partner and anneal to form duplexes. If the two DNAs have
significant sequence similarity, they also tend to form partial duplexes or
hybrids with each other. The greater the sequence similarity between the two
DNAs, the greater the number of hybrids formed

Know proteins and their interactions

Proteins are organic compounds made of amino acids arranged in a linear
chain and folded into a globular form
The amino acids in a polymer are joined together by the peptide bonds
between the carboxyl and amino groups of adjacent amino acid residues
The sequence of amino acids in a protein is defined by the sequence of a
gene, which is encoded in the genetic code
Protein-protein interactions involve not only the direct-contact
association of protein molecules, but also long range interactions through the
electrolyte, aqueous solution medium surrounding neighbor hydrated proteins
o The interactions between proteins are important for most of the
biological functions
Know pH, pI, pK
pH (potential for hydrogen ion concentration) is a measure of the
acidity or basicity of a solution
pI (isoelectric point, isoelectric pH) is the characteristic pH that which
the net electric charge is 0

pK (acid dissociation constant) is a quantitative measure of the strength

of an acid in solution. It is the equilibrium constant for a chemical reaction
known as dissociation in the context of acid-base reactions

Hemoglobin versus Myoglobin

Hemoglobin is the oxygen-carrying protein of erythrocytes (red blood cells)
o The oxygen transporting capacity is dependent on four iron ions
Myoglobin is an oxygen-binding protein found in the muscle tissue
It is the heme portion of iron that gives both red blood cells and red muscle
their color
In blood, it is the hemoglobin that contains iron, and in muscle cells, the
myoglobin contains the iron
Red blood cells contain hemoglobin, not myoglobin, and so it is hemoglobin
that gives red blood cells their color
Know intermolecular forces
Intermolecular forces are forces that act between stable molecules or
between functional groups of macromolecules
o includes momentary attractions between molecules, diatomic free
elements, and individual atoms
o these forces, notably London dispersion forces, dipole-dipole
interactions, and hydrogen bonding, are significantly weaker than
either ionic or covalent bonding, but still have a noticeable chemical
effect
o intermolecular forces are due to differences in charge density in
molecules
Know about amino acid side chains
Amino acids are molecules containing an amine group, a carboxylic acid
group, and a side chain that varies between different amino acids
o These side chains can vary in size from just a hydrogen atom in
glycine, to a methyl group in alanine, through to a large heterocyclic
group in tryptophan
o Amino acids are usually classified by the properties of their side chains
into four groups
The side chain can make an amino acid a weak acid, or a weak
base, and a hydrophile if the side chain is polar, or a hydrophobe
if it is nonpolar
Know about the techniques of separating proteins
Column chromatography takes advantage of differences in protein charge,
size, binding affinity, and other properties
Cation-exchange chromatography, the solid matrix has negatively
charged groups. In the mobile phase, proteins with a net positive charge
migrate through the matrix slowly than those with a net negative charge,
because the migration of the former is retarded more by interaction with the
stationary phase

Size-exclusion chromatography separates proteins according to size. In

this method, large proteins emerge from the column sooner than small ones
a somewhat counterintuitive result
o The solid phase consists of beads with engineered pores or cavities of
a particular size
o Large proteins cannot enter the cavities, and so take a short and rapid
path through the column, around the beads
o Small proteins enter the cavities and migrate through the column more
slowly
Affinity chromatography is based on the binding affinity of a protein. The
beads in the column have a covalently attached chemical group. A protein
with affinity for this particular chemical group will bind to the beads in the
column, and its migration will be retarded as a result
HLPC (high-performance liquid chromatography) makes use of highpressure pumps that speed the movement of the protein molecules down the
column down the column, as well as higher-quality chromatographic
materials that can withstand the crushing force of the pressurized flow
o HLPC can limit diffusional spreading of protein bands and thus greatly
improve resolution

Know about the NMR

Nuclear Magnetic Resonance (NMR) is a method for determining the
three-dimensional structures of macromolecules
o Its the property that magnetic nuclei have in a magnetic field that
applied electromagnetic pulse or pulses, which cause the nuclei to
absorb energy from the EM pulse and radiate this energy back out
o The energy radiated back out is at a specific resonance frequency
which depends on the strength of the magnetic field and other factors
Know about the mass spectrometer
Mass spectrometry is an analytical technique for the determination of the
elemental composition of a sample or molecule
o The MS principle consists of ionizing chemical compounds to generate
charged molecules or molecule fragments and measurement of their
mass-to-charge ratios
Know X-ray crystallography
X-ray crystallography is a method of determining the arrangement of
atoms within a crystal, in which a beam of X-rays strikes a crystal and
diffracts into many specific directions
o From the angles and intensities of these diffracted beams, a
crystallographer can produce a three-dimensional picture of the
density of electrons within the crystal
o From this electron density, the mean positions of the atoms in the
crystal can be determined, as well as their chemical bonds, their
disorder and various other information
Fate of pyruvate

Pyruvate is a molecule that is a key intersection in several metabolic

pathways
o It can be made from glucose through glycolysis, supplies energy to
living cells in the citric acid cycle, and can also be converted to
carbohydrates via gluconeogenesis, to fatty acids or energy through
acetyl-CoA, to the amino acid alanine and to ethanol

Know what anaerobic and aerobic conditions are

Anaerobic conditions are virtually devoid of oxygen, and microorganisms
adapted to these environments obtain energy by transferring electrons to
nitrate (forming N2), sulfate (forming H2S), or CO2 (forming CH4)
Aerobic conditions have a plentiful supply of oxygen, and some resident
organisms derive energy from the transfer of electrons from fuel molecules to
oxygen
Know regulation of gluconeogenesis
While most steps in gluconeogenesis are the reverse of those found in
glycolysis, three regulated and strongly exergonic reactions are replaced with
more kinetically favorable reactions
o Hexokinase/glucokinase, phosphofructokinase, and pyruvate kinase
enzymes of glycolysis are replaced with glucose-6-phosphate, fructose
1,6-biphosphatase, and PEP carboxykinase
o The majority of the enzymes responsible for gluconeogenesis are found
in the cytoplasm
o The rate of gluconeogenesis is ultimately controlled by the action of a
key enzyme, fructose 1,6-biphosphatase, which is also regulated
through signal transduction by cAMP and its phosphorylation
o Acetyl CoA and citrate activate gluconeogenesis enzymes (pyruvate
carboxylase and fructose 1,6-biphosphatase, respectively)
Due to the reciprocal control of the cycle, acetyl-CoA and citrate
also have inhibitory roles in the activity of pyruvate kinase

Know regulation of the citric acid cycle

The regulation of the TCA cycle is largely determined by substrate availability
and product inhibition
NADH, a product of all dehydrogenasees in the TCA cycle (exception:
succinate dehydrogenase) inhibits pyruvate dehydrogenase, isocitrate
dehydrogenase, -ketoglutarate dehydrogenase, and also citrate synthase
Acetyl CoA inhibits pyruvate dehydrogenase
Succinyl-CoA inhibits succinyl CoAsynthease and citrate synthase

ATP inhibits citrate synthase and a-ketoglutarate dehydrogenase

Calcium is used as a regulator, and it activates pyruvate dehydrogenase,
isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase
Citrate is used for feedback inhibition, as it inhibits phospofructokinase, an
enzyme involved in glycolysis, that catalyzes formation of fructose 1,6biphosphate, a precursor of pyruvate

Know regulation of the glycolysis

Glycolysis is regulated by slowing down or speeding up certain steps in the
glycolysis pathway

o
o
o

This is accomplished by inhibiting or activating the enzymes that are

involved
Any step with a free energy near 0 is not being regulated
A step with a large negative change in free energy is assumed to be
regulated

Know the pyruvate dehydrogenase complex

The pyruvate dehydrogenase complex is a complex of three enzymes
that transform pyruvate into acetyl-CoA by a process called pyruvate
decarboxylation
o Acetyl-CoA may then be used in the citric acid cycle to carry out
cellular respiration, and this complex links the glycolysis metabolic
pathway to the citric acid cycle
Pyruvate decarboxylation is also known as the pyruvate dehydrogenase
reaction because it also involves the oxidation of pyruvate
Know signal transduction
Signal transduction is the conversion of information into a chemical change
o The signal represents information that is detected by specific receptors
and converted to a cellular response, which always involves a chemical
process
Know secondary messengers
Second messengers are molecules that relay signals from receptors on the
cell surface to target molecules inside the cell, in the cytoplasm, or in the
nucleus
o They relay the signals of hormones, growth factors, and others, and
cause some kind of change in the activity of the cell
o They greatly amplify the strength of the signal
o Secondary messengers are a component of signal transduction
cascades
Know the function of the receptor tyrosine kinase (RTK)
Receptor tyrosine kinases (RTK) are the high-affinity cell surface
receptors for many polypeptide growth factors, cytokines, and hormones
o Shown not only to be key regulators of normal cellular processes, but
also have a critical role in the development and progression of many
types of cancer
Know the pentose phosphate pathway
The pentose phosphate pathway leads to the oxidation of glucose 6phosphate to pentose phosphates
o Its a process that generates NADPH and pentoses
o There are two distinct phases in the pathway
The first is the oxidative phase, in which NADPH is generated,
and the second is the non-oxidative synthesis of 5-carbon sugars
This pathway is an alternative to glycolysis

Recognize enzymes of the citric acid cycle

See the above diagram
Know the difference between catabolic and anabolic pathways
Catabolic pathways degrade organic nutrients into simple end products in
order to extract chemical energy and convert it into a form useful to the cell
Anabolic pathways start with small precursor molecules and convert them
to progressively larger and more complex
Know transition state analogs
Transition state analogs are chemical compounds with a chemical
structure that resembles the transition state of a substrate molecule in an
enzyme-catalyzed chemical reaction
o Transition state analogs do not undergo a chemical reaction and can
act as enzyme inhibitors by blocking their active site
Know about serine proteases
Serine proteases are proteases (enzymes that cut peptide bonds in
proteins) in which one of the amino acids at the active site is serine
Know what chymotrypsin is
Chymotrypsin is a digestive enzyme that can perform proteolysis (directed
degradation/digestion of proteins by cellular enzymes called proteases or by
intramolecular digestion)

Preferentially cleaves peptide amide bonds where the carboxyl side of

amide bond is a tyrosine, tryptophan, or phenylalanine
They contain an aromatic ring in their side chain that fits into a
hydrophobic pocket of the enzyme

Whats a buffer solution?

A buffer solution is an aqueous solution consisting of a mixture of a weak
acid and its conjugate base or a weak base and its conjugate acid
Whats equilibrium?
When a system is at equilibrium, the rate of product formation exactly
equals the rate at which product is converted to reactant
o There is no net change in the concentration of reactants and products;
a steady state is achieved
Whats phosphorylation?
Phosphorylation involves the transfer of phosphoryl groups
The purpose of this has to do with conformational change
Phosphorylation is the addition of a phosphate group to a protein or other
organic molecule
o Phosphorylation activates or deactivates many protein enzymes
Whats the electron transport chain?
An electron transport chain couples a chemical reaction between an
electron donor (such as NADH) and an electron acceptor (such as O2) to the
transfer of H ions across a membrane, through a set of mediating biochemical
reactions
o The H ions are used to produce adenosine triphosphate (ATP), the main
intermediate in living organisms as they move back across the
membrane
o ETC are used for extracting energy from sunlight (photosynthesis) and
from redox reactions such as the oxidation of sugars (respiration)