Prokaryotic:

Simplest cells

Organisms that lack a nuclear membrane

Unicellular organisms that do not develop or differentiate into multicellular forms.
Three architectural regions: appendages called flagella and pili (proteins attached to the cell
surface); a cell envelope consisting of a capsule, a cell wall and a plasma membrane; and a
cytoplasmic region that contains the cell genome and ribosomes and various sorts of
inclusions.

Eukaryotic:

Contain membrane-bounded compartments in which specific metabolic activities take place.

A nucleus, a membrane-delineated compartment that houses the eukaryotic cell's
deoxyribonucleic acid (DNA)
Other specialized structures, called organelles, which are small structures within cells that
perform dedicated functions.

Cell differentiation: (add pic)

All the different cell types in our body are all derived from a single, fertilized egg cell through
differentiation.
Differentiation is the process by which an unspecialized cell becomes specialized into one of
the many cells that make up the body, such as a heart, liver or muscle cell.
During differentiation, certain genes are turned on, or become activated, while other genes
are switched off, or inactivated. This process is intricately regulated. As a result, a
differentiated cell will develop specific structures and perform certain functions.

Central dogma: ( pic)

The Central Dogma, states that genetic information flows from DNA to RNA to protein. The genetic
code resides in DNA is passed from generation to generation.

DNA transcription refers to the synthesis of RNA from a DNA template.

The most important enzyme is RNA polymerase. It is an enzyme that influences the synthesis of RNA
from a DNA template. In order for transcription to be initiated, RNA polymerase must be able to
recognize the beginning sequence of a gene so that it knows where to start synthesizing an mRNA. It
is directed to this initiation site by the ability of one of its subunits to recognize a specific DNA
sequence found at the beginning of a gene called the promoter sequence.

The RNA polymerase then unwinds the double helix at that point and begins synthesis of an RNA
strand complementary to one of the strands of DNA. This strand is called the antisense or template
strand, while the other strand is referred to as the sense or coding strand. Synthesis can then
proceed in a unidirectional.

The coding portions of a gene, called exons, are interrupted by intervening sequences, called introns.

Both exons and introns are transcribed into mRNA, but before it is transported to the ribosome, the
primary mRNA transcript is edited. This editing process removes the introns, joins the exons
together, and adds unique features to each end of the transcript to make a mature mRNA.

The cellular machinery responsible for synthesizing proteins is the ribosome. The ribosome consists
of structural RNA and about 80 different proteins. In its inactive state, it exists as two subunits: a
large subunit and a small subunit. When the small subunit encounters an mRNA, the process of
translating an mRNA to a protein begins. In the large subunit, there are two sites for amino acids to
bind, and thus be close enough to each other to form a bond. The "A site" accepts a new transfer
RNA, or tRNA, which bears an amino acid and is the adaptor molecule acting as a translator between
mRNA and protein. The "P site" binds the tRNA that becomes attached to the growing chain.
The tRNA is a specific RNA molecule. Each tRNA has a specific acceptor site that binds a particular
triplet of nucleotides, codon, and an anticodon site that binds a sequence of three unpaired
nucleotides, the anticodon, which can then bind to the codon. Each tRNA also has a specific charger
protein; called an aminoacyl tRNA synthetase. This protein can only bind to that particular tRNA and
attach the correct amino acid to the acceptor site. Each codon specifies a particular amino acid. In
this way, the ribosomal complex builds a protein one amino acid at a time, with the order of amino
acids determined precisely by the order of the codons in the mRNA.

U
UUU
u Phenylalanine
UUC
Phenylalanine
UUA
CUU
Leucine
Leucine UUG
CUC
c Leucine
Leucine
CUA
Leucine CUG
Leucine
AUU
Isoleucine AUC
A Isoleucine AUA
Isoleucine AUG
Methionine
GUU
Valine GUC
G
Valine QUA
Valine GUG
Valine

C
A
UCU
UAU
Serine UCC
Tyrosine UAC
Serine UCA
Tyrosine UAA
Serine UCG
Stop UAG
Serine
Stop
CCU
CAU
Proline CCC
Histidine CAC
Proline CCA
Histidine CAA
Proline CCG
Glutamine CAG
Proline
Glutamine
ACU
AAU
Threonine ACC Asparagine AAC
Threonine ACA Asparagine AAA
Threonine ACG
Lysine AAG
Threonine
Lysine
GCU
GAU
Alanine GCC
Aspartate GAC
Alanine GCA
Aspartate GAA
Alanine GCG
Glutamate GAG
Alanine
Glutamate

G
UGU
Cysteine UGC
Cysteine UGA
Stop UGG
Tryptophan
CGU
Arginine CGC
Arginine CGA
Arginine CGG
Arginine
AGU
Serineine AGC
Serineine AGA
Arginine AGG
Arginine
GGU
Glycine GGC
Glycine GGA
Glycine GGG
Glycine

DNA Isolation:

DNA isolation refers to the process of extracting DNA from a cell in a relatively pure form. It involves
separating DNA from other cellular components, such as proteins, RNA, and lipids.

The cells used to obtain and isolate the DNA could come directly from tissue, or could be cultured
laboratory cell lines obtained using the methods described earlier.
Whatever the source, the DNA is isolated by placing the cells in a tube containing a special solution
called a "cocktail" and mechanically or chemically breaking them open. This causes the cell to
release its contents into the cocktail containing enzymes, chemicals, and salts.
Enzymes are used to chew up the proteins; chemicals are used to destroy any RNA present; and salts
are used to help pull the DNA out of solution.
At this point, the DNA will exist in long strands that form a mucous-like glob within solution.
The DNA is then harvested by spinning the tube in a machine called a centrifuge. During spinning,
the DNA collects in the bottom of the tube.

The solution is then poured off, and the DNA is dissolved, or re-suspended, in a second solution that
will make it easy to work with in subsequent procedures.

The result is a concentrated DNA sample containing many thousands of copies of each gene. For
large-scale DNA analysis methods, such as those required to sequence the human genome, DNA
isolation is performed using robots.
The primary reagents used in PCR:
The primary materials, or reagents, used in PCR are:
DNA nucleotides - the building blocks for the new DNA
Template DNA - the DNA sequence that you want to amplify
Primers - single-stranded DNAs between 20 and 50 nucleotides long that are complimentary to a
short region on either side of the template DNA
Tag polymerase - a heat stable enzyme that drives, or catalyzes, the synthesis of new DNA
An algorithm and A program

The difference between an algorithm and a program: the former is a set of steps that define some
computational process at an abstract level; the latter is the implementation of an algorithm. There

may be many different implementations of the same algorithm, but these should give the same
results, if the algorithm has been clearly define.

BLAST.

BLAST (Basic Local Alignment Search Tool) provides a method for rapid searching of nucleotide and
protein databases. Since the BLAST algorithm detects local as well as global alignments, regions of
similarity embedded in otherwise unrelated proteins could be detected. Both types of similarity may
provide important clues to the function of uncharacterized proteins.

Restriction Enzymes
Restriction Enzymes Make Sequence-Specific Cuts in DNA:

Problem: Natural DNA is tangled spaghetti that is too long to manage.

Average size of human chromosome ~ 130,000,000bp.

~ 3 billion bp in the human genome

Solution: Use Restriction Enzymes to cut DNA into fragments.

Phenomena: When one strain of E. Coli would infect another, its DNA would be fragmented
into pieces. (1953)

In rare cases the infecting DNA molecule would not be broken down.

The first restriction enzyme was identified in H. Influenza in 1970.

Restriction enzyme: A defence mechanism produced in bacteria.

Acts like scissors, cuts DNA at specific cleavage site.

The cleavage site: target sequence 4-8bps long.

In many cases the target is palindrome.


A DNA locus whose 5'-to-3' sequence is identical on each DNA strand. The sequence is the
same when one strand is read left to right and the other strand is read right to left. Recognition sites
of many restriction enzymes are palindromic.

All together over 150 different restriction enzymes have been found.

Many cuts are staggered and leave sticky ends. Ends on one or more DNA molecules that
have short, overhanging, single-stranded, complimentary segments that help the ends bind to each
other.

All the cleavage sites will be cut in a process called digestion.

By using cleavage sites of different sizes we can cut the DNA into fragments of different
lengths

Fragment analysis by gel electrophoresis

Gel Electrophoresis:
1. The DNA fragments produced by a restriction enzyme can be separated by size using the fact that
DNA is negatively charged and moves in response to an electric field.
2. If the terminals of an electrical power source are connected to the opposite ends of a horizontal
tube containing a DNA solution, then the DNA molecules will move toward the positive end of the
tube at a rate that depends on the electric field strength and on the shape and size of the molecules.
The movement of charged molecules in an electric field is called electrophoresis.
3. A thin slab of a gel, usually agarose or acrylamide, is prepared containing small slots (called wells)
into which, samples are placed.
4. An electric field is applied, and the negatively charged DNA molecules penetrate and move
through the gel toward the anode (the positively charged electrode).
5. Hence the rate of movement increases as the size of the DNA fragment decreases.
6. The result of electrophoresis of a set of double-stranded DNA molecules in an agarose gel. Each
discrete region containing DNA is called a band. The bands can be visualized under ultraviolet light
after soaking the gel in the dye ethidium bromide, the molecules of which intercalate between the
stacked bases in duplex DNA and render it fluorescent.

Ethidium Bromide

DNA can absorb UV light (Hyperchromic Shift), however this absorption is not sufficiently sensitive.

Staining is necessary and the most common dye used for visualizing nucleic acids is ethidium
bromide.

The structural similarity between ethidium and paired bases in DNA makes it possible for the
ethidium molecule to intercalate in between the base pairs stacked down the center of the double
helix and render the DNA fluorescent.

Molecular Biology Workflow

Molecular biologist typically utilizes a series of technique to solve a biological question

Usually the biological problem can be broken down into multiple smaller sub-problems

For example, to search for a gene involved in neural degeneration in human, one may break
this problem into many parts

Show that the candidate gene does exist in human using low stringency hybridization
method

Screen the human DNA library using the hybridization conditions established in the first step

The first part (show that the candidate gene does exist in human using low stringency
hybridization method) involves many established molecular biology techniques that are typically
carried out in a sequence or workflow

Steps

Preparation of DNA used for probe by PCR

Purification of PCR DNA by agarose gel electrophoresis

Radioactive labeling of DNA probe

Southern analysis


Molecular biology workflow consists of a sequence of techniques designed to satisfy an
experimental objective

Restriction enzymes like hind ll break DNA at the center of their recognition sites to produce blunt
fragment that are base-paired out to their ends and have no tendency to stick together.

The EcoRi enzymes makes staggered cuts that create short four-based single stranded tails on the
ends of each fragment. Many other restriction enzyme also make staggered cuts, leaving singlestranded tail sequences specific for each enzyme. Complementary single-stranded tails tend to
associate by base pairing and thus are often called cohesive, or have sticky end.