Download Introduction to DNA

Introduction to DNA
Chapter 4
4.1 DNA Structure and Function
Manipulations of DNA
and RNA are center for
biotech research
“Central Dogma”
Small sections of
Transcription
DNA (genes)
mRNA
Translations
at ribosome
proteins
4.1 DNA Structure and Function
Pyrimidines
Purines
4.1 DNA Structure and Function
Similarities in DNA Molec. Among
Organisms
Made of 4 nucleotide monomers
that contain 4 nitrogenous bases
Form double helix (2 sides) of
repeating nucleotides, several
million pb in length ie…same
DNA structure
A from one strand always with T
from another and C from one
strand always with G from
another and ….so equal amounts
of each
A,T, C, G are stacked 0.34 nm
apart with 10 nitrogen bases per
complete turn of the helix
Variations in DNA Molec.
Number of DNA strands in the
cells of an organism (i.e. # of
chromosomes)
Length in bp of DNA strand
Number and types of genes
Shape of DNA strand (circular
or linear chromosomes)
4.2 Sources of DNA
Prokaryotic
Bacterial cell culture
Eukaryotic
Mammalian cell culture
Viral
4.2 Prokaryotic DNA
Bacteria cells ex E.Coli
Do not contain nucleus or membrane bound organelle
DNA is floating in cytoplasm
Typical bacterium contain only one circular long DNA (a
chromosome)
Some bacteria have extra small rings of DNA (plasmids)
4.2 Prokaryotic DNA
Most familiar plasmids are R plasmids
Contain antibiotic resistance genes
Bacteria with these genes can survive antibiotics
that would normally kill them
Bacteria can transfer plasmids ie genetic info
between themselves
So, genes for antibiotic resistance can be
transferred btw bacteria and lead to deadly
antibiotic resistant disease causing bacteria
Gives bacteria a way of evolving
4.2 Prokaryotic DNA
Different bacteria have different plasmid
Some bacteria have more than one kind
Some bacteria have none
Plasmids are small and easy to extract
from cells
Often used as rDNA vectors to transform cells
4.2 Prokaryotic DNA
DNA fragments (genes) can be cut and
pasted into plasmid vector
Recombinant plasmid can then get introduced
to cell
Cell will read DNA code on the r-plasmid
Then start synthesizing proteins coded for the
gene
Same procedures Genentech, Inc used to
produce human insulin from E-Coli
Gene Expression Process
Prokaryotic DNA’s gene expression (genes
turned on or off ) is simple with only few
controls
An operon (1 or more genes and their controlling
elements)
RNA polymerase (enzyme that synthesize mRNA
molec.) attach to DNA segment at a promoter region
of operon
this “turns on” gene
RNA polymerase works its way down DNA strand
to structural gene to built mRNA
mRNA is decoded into a peptide at a ribosome
Operator can then “turn off” the gene
Operon
Section that codes for mRNA
which later get translated to
proteins
Gene Expression Process
In case of regulatory molec. Attach at operon
Operon get “turned off” b/c RNA polymerase is blocked from
continuing down the strand to the gene
No protein is produced
Blocking and unblocking is how bacteria make certain proteins
certain times
Example: Lactose
Bacterial Cell Culture
To manipulate bacteria DNA, cells are
needed
Some bacteria prefer
Liquid medium (Broth)
Solid medium (agar)
Grow well in either
Agar
Mixture of water and protein molec.
Preparation:
Powdered agar and water are mixed, heated
until agar completely suspended
Agar is sterilized at high temps (121C or
higher) and high pressure (15 psi or higher)
cooled to 65C and poured under sterile
condition in Petri dishes
Agar cools and solidifies in 15-20 min
Plates can be used after 24 hr.
Broth
Also water and protein molec.
Cultures grow as suspension of million of
floating cells
4.2 Eukaryotic DNA
From protist, fungi, plants, and animal cells
Same as prokaryotic
Same nucleotides (A, C, G, T)
Same double helix of repeating nucleotides
Each antiparallel strand bound to the other by H-bonds
Different from prokaryotic
Packed into chromosomes (regulated and expressed different
from bacteria)
Several chromosomes/cell vs. only 1/cell
Each chromosome is single, linear, very long molec of DNA
Lack of operators
Chromosomes range 4-100 or more
Human have 46 chromosomes, fruit flies 8, ferns more than 1000
Amount of DNA/cell is not directly related to organism’s
complexity
Gene Expression
Controlled differently than prokaryotes
Eukaryotic gene expressed at very low level
Increase in expression happen when enhancer
(section of DNA) molec. interact with RNA
polymerase or with enhancer DNA regions
Molecules attach to DNA and increase gene
transcription
Gene Expression
Gene contains promoter region where RNA molec. recognize the
gene
RNA polymerase attach and move down DNA molec. To get to
structural genes
At structural genes RNA polymerase builds a complementary
mRNA transcript from one side of DNA strand
The enzyme transcribes the entire gene until reaches a termination
sequence
Gene Expression
Prokaryotic
mRNA transcript is
immediately translated
into polypeptide at a
ribosome
No introns and exons at
structural genes
Operator to regulate
gene expression
Eukaryotic
mRNA is often modified before
translation
Structural genes are made of
intron and exon sections
Exons: DNA sections that contain
the protein code
Introns: spacer DNA
No operator so produce molec.
called transcription factors to turn
genes on
Also regulated by way
chromosome coiled (around
histones proteins)
Coiling makes genes buried and
RNA polymerase can not get to
them
DNA has to uncoil
Mammalian Cell Culture
Growing them more challenging that
bacteria
Normally grown in broth culture
4.2 Eukaryotic DNA
Viral DNA
Viruses
Do not have cellular structure
Collection of protein and nucleic acid molec.
that become active once they are within a
suitable cell
Very small, measure from 25-250 nm
Based on type of cell they attach classified as:
Bacterial, plant, or animals
Have thick protein coat surrounding nucleic
acid core of either DNA or RNA
Animal virus
Viral DNA
Within a cell:
Nucleic acid of a virus is released
Viral genes are read by host cell enzyme
Decoded into viral mRNA
Translated into viral proteins
New virus molec. are assembled and released
and may infect other cells
Viral DNA
Viral DNA and RNA molec are short, easy
to manipulate
Called vectors
4.3 Isolating and Manipulating
DNA
Genetic engineering (G.E.):
All modifications of DNA code of an
organism
Process:
ID of molec produced by living things that could
be produced more economically or easily through
G.E. (ex. insulin)
Isolation of gene for production of molec (insulin
gene)
Manipulation of DNA instruction
Harvesting of molecule or product
Recombinant DNA Technology
Methods used to create new DNA molec
by piecing together different DNA molec
When cells accept rDNA and start expressing
the new genes by making new proteins….they
are considered genetically engineered
Many items on market with rDNA
technology
Ex recombinant rennin , chymosin
Site-Specific Mutagenesis
Process of inducing changes in certain sections
of a particular DNA code
Accomplished through use of chemicals,
radiation, viruses
Sometimes “directed”(on purpose by scientist to
make better changes)
Ex: subtilisin marketed by Genecor international
(tide)
An enzyme (protease) that degrades proteins
Added to laundry detergent to stains like blood,
gravy
Gene Therapy
Process of correcting faulty DNA codes that
causes genetic diseases and disorders
Common way to use virus to carry a normal
gene into cells containing defective ones (gene
replacement)
Correct diseases like Parkinson's, diabetes, cystic
fibrosis (CF), and some cancers
CF:
One of every 3000 babies born with it
Causes build up of thick mucus that clogs the
respiratory and digestive systems
Gene Therapy
In 2002:
Modified cold virus used to transfer normal copy of
the gene cystic fibrosis transmembrane conductance
regulator (CFTR)
To cells lining the nose
CFTR gene is defective in CF patients
Regulates flow of Cl- ions into epithelial cells lining
respiratory and digestive systems
This method called adeno-assosiated virus (AAV)
delivery system b/c uses modified adenovirus (cold
virus)
Worked only in small # still in clinical trials
4.4 Gel Electrophoresis
Uses electricity to separate charged molec. on a
gel slab
Separation based on size, shape and charge
Ex: DNA and RNA fragments, and proteins
Gel:
Powdered agarose (carb. derived from seaweed)
Dissolve in boiling buffer soln.
Most common agarose is polyacrylamide (PAGE)
Gel solidify and placed in get box and covered with
buffer soln.
Gel Electrophoresis
Gel Electrophoresis
Gel Electrophoresis
Agarose used when separating
DNA pieces no smaller than 500bp and no
larger than 25,000bp
Made at a specific concentrations ranging (0.63%) figure 4.32
High concentration for smaller molec
Gel Electrophoresis
Gel stains:
Nucleic acids are colorless
Must be stained
DNA stains:
Ethidium bromide (EtBr)…orange when mixed
with DNA under UV light
Methylene blue…dark blue…not as sensitive as
EtBr viewed with white light
Gel Electrophoresis
most common
Sizing standard
Only one DNA type
Plasmid restriction digestion
DNA sample from bacterial chromosome
RNA
Note: Only
negative charge
would run on
this one
Smears (thousands
of different size
molec in small
concentration)
No nucleic acids
DNA so large
will not load
Ex: eukaryotic
genome
Homework
Sec 4.3 Review question 1, 2, 3, 4
Sec 4.2 Review questions 1, 2, 3, 4
Sec 4.4 Review questions 2, 3, 4
Think like Biotech
2, 3, 6, 7, 8
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