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FE314- Biotechnology
Spring 2017
Lecture 2
Recombinant DNA Technology and Biotechnology
What is recombinant DNA ?
• Technique of manipulating the genome of a
cell or organism so as to change the
phenotype desirably.
Seedless guava
Calorie free sugar
DNA structure
#1. DNA Structure (an overview)
• DNA has three main components
– 1. deoxyribose (a pentose sugar)
– 2. base (there are four different ones)
– 3. phosphate
#2. The Bases
 They are divided into two groups
 Pyrimidines and purines
 Pyrimidines (made of one 6 member ring)
 Thymine
 Cytosine
 Purines (made of a 6 member ring, fused to a 5
member ring)
 Adenine
 Guanine
 The rings are not only made of carbon (specific
formulas and structures are not required for IB)
#3. Nucleotide Structure
• Nucleotides are formed by the condensation
of a pentose sugar, phosphate and one of the
4 bases
• The following illustration represents one
nucleotide
#3. Nucleotide Structure
• Nucleotides are linked together by covalent
bonds called phosphodiester linkage
#4. DNA Double Helix and Hydrogen Bonding
• Made of two strands of nucleotides that are
joined together by hydrogen bonding
• Hydrogen bonding occurs as a result of
complimentary base pairing
– Adenine and thymine pair up
– Cytosine and guanine pair up
– Each pair is connected through hydrogen bonding
– Hydrogen bonding always occurs between one
pyrimidine and one purine
#4. DNA Double Helix and Hydrogen Bonding
• Complimentary base pairing of pyrimidines
and purines
#4. DNA Double Helix and Hydrogen Bonding
#1. DNA Structure (an overview)
• DNA has three main components
– 1. deoxyribose (a pentose sugar)
– 2. base (there are four different ones)
– 3. phosphate
#2. The Bases
 They are divided into two groups
 Pyrimidines and purines
 Pyrimidines (made of one 6 member ring)
 Thymine
 Cytosine
 Purines (made of a 6 member ring, fused to a 5
member ring)
 Adenine
 Guanine
 The rings are not only made of carbon (specific
formulas and structures are not required for IB)
#3. Nucleotide Structure
• Nucleotides are formed by the condensation
of a pentose sugar, phosphate and one of the
4 bases
• The following illustration represents one
nucleotide
#3. Nucleotide Structure
• Nucleotides are linked together by covalent
bonds called phosphodiester linkage
#4. DNA Double Helix and Hydrogen Bonding
• Made of two strands of nucleotides that are
joined together by hydrogen bonding
• Hydrogen bonding occurs as a result of
complimentary base pairing
– Adenine and thymine pair up
– Cytosine and guanine pair up
– Each pair is connected through hydrogen bonding
– Hydrogen bonding always occurs between one
pyrimidine and one purine
#4. DNA Double Helix and Hydrogen Bonding
• Complimentary base pairing of pyrimidines
and purines
#4. DNA Double Helix and Hydrogen Bonding
#4. DNA Double Helix and Hydrogen Bonding
•Adenine always pairs
with thymine because
they form two H bonds
with each other
•Cytosine always pairs
with guanine because
they form three
hydrogen bonds with
each other
#5. DNA Double Helix
• The ‘backbones’ of DNA molecules are made
of alternating sugar and phosphates
• The ‘rungs on the ladder’ are made of bases
that are hydrogen bonded to each other
#6. Antiparallel strands
5’
3’
The strands
run opposite
of each
other.
The 5’ end
always has
the
phosphate
attached.
3’
5’
Assignment (in your notebook)
• 1. Draw the structure of ribose and number the carbons
• 2. Draw a schematic representation of a nucleotide. Label
the sugar, base and phosphate.
• 3. What are the complimentary base pairs to a DNA strand
that has the following order A T A C C T G A A T?
• 4. Draw a schematic representation of an unwound DNA
double helix using the base pairs from your answer in
question 3.
– Include the number of hydrogen bonds between each base
pair. Be sure to label all of the bases and the 5’ and 3’ ends of
the structure.
#6. When phosphodiester links are
formed . . .
• A. When the covalent bonds are formed
between nucleotides the attach in the
direction of 5’→3’
• B. The 5’ end of one nucleotide attaches to
the 3’ end of the previous nucleotide
#7. Nucleosome structure
• Nucleosome are the basic unit of chromatin
organization
• In eukaryotes DNA is associated with proteins
– (in prokaryotes the DNA is naked)
• Nucleosomes = basic beadlike unit of DNA packing
– Made of a segment of DNA wound around a
protein core that is composed of 2 copies of each
of 4 types of histones
#8. Genes
• Genes=units of genetic information (hereditary
information)
• Order of nucleotides make up the genetic code
• Genes can contain the information for one polypeptide
• Genes can also regulate how other genes are expressed
• All cells of an organism contain the same genetic
information but they do not all express the same genes
– THIS IS CELL DIFFERENTIATION
– Cells differentiate by genes that are activated
#8. Genes
• Repetitive sequences-part of the non-coding
section of DNA
– Function-unknown
– Can be used in DNA profiling (DNA fingerprinting)
Basic steps involved in process
Introducing in
Host
Culturing the
cells
Isolating
genomic DNA
Insertion of
DNA in a
vector
Fragmenting
this DNA
Transformation of
host cell
Screening
the
fragments
Basic steps involved in process
Isolating
genomic DNA
Fragmenting
this DNA
Isolating
genomic DNA
from the donor.
Fragmenting
this DNA using
molecular
scissors.
Basic steps involved in process
Screening
the
fragments
Insertion of
DNA in a
vector
Screening the
fragments for a
“desired gene”.
Inserting the
fragments with the
desired gene in a
‘cloning vector’.
Basic steps involved in process
Introducing
in Host
Introducing the recombinant
vector into a competent host
cell
Culturing
the cells
Culturing these cells to obtain
multiple copies or clones of
desired DNA fragments
Transformation
of host cell
Using these copies to
transform suitable host cells
so as to express the desired
gene.
Tools used in recombinant DNA
technology
• Enzymes
• Vectors
Tools used in recombinant DNA
technology
• Enzymes
Act as biological scissors.
Most commonly used are:
 Restriction endonuclease
DNA ligase
DNA polymerase
Alkaline phosphatases
Tools used in recombinant DNA
technology
• Vectors
Low molecular weight DNA
molecules.
Transfer genetic material into
another cell.
Capable of multiplying
independently.
Vector
Bacteriophag
e DNA
Plasmi
d
Vector
Cosmid
Artificia
l DNA
Insertion of vector in target cell is
called
• Bacterial cells – Transformation
• Eukaryotic cells – Transfection
• Viruses - Transduction
Insertion of vector in target cell
Vectors used:
•
•
•
•
Bacteria- plasmids, cosmid, lambda phage
Insects- baculoviruses
Plants- Ti plasmid
Yeast cells- YAC (yeast artificial chromosome)
HOST
DONOR
DNA
DNA
Fragmented by Restriction Endonuclease
DNA strands with sticky ends
Sticky ends base pair with complementary sticky ends
DNA ligase links them to form rDNA
In vitro
Polymerase chain
reaction (PCR)
Cloned
In vivo
Prokaryotic or eukaryotic
cell, mammalian tissue
culture cell
Some examples of therapeutic
products made by recombinant DNA
techniques
¶ Blood Proteins: Erythropoietin, Factors VII, VIII, IX;
Tissue plasminogen activator; Urokinase.
¶ Human Hormones: Epidermal growth factor; Follicle
stimulating hormone, Insulin.
¶ Immune Modulators: α Interferon, β Interferon;
Colony stimulating hormone; Lysozyme; Tumor
Necrosis factor.
¶ Vaccines: Cytomegalovirus; Hepatitis B; Measles;
Rabies
Transposons
• Transposons are sequences of
DNA that can move or transpose
themselves to new positions
within the genome of a single
cell.
• Also called ‘Jumping genes’.
• 1st transposons were discovered
by
Barbara
McClintock
in Zea mays (maize)
Types of transposons
• According to their mechanism
they are classified as:
Retrotransposons
• Follows method of “Copy and
Paste”.
• Copy in two stages.
DNA
RNA
Transcription
DNA
Reverse
Transcription
DNA transposons
• Follows the method of “Cut and
Paste”.
• Do not involve RNA
intermediate.
Enzyme Transposase
Cuts out transposon
Ligates in new position
Plasmid
• Plasmids are small, extra chromosomal,
double stranded, circular forms of DNA that
replicate autonomously.
• The term was introduced by
in
1952.
Joshua
Lederberg
Plasmid
• Found in bacterial, yeast and occasionally in
plants and animal cells.
• Transferable genetic elements or
‘Replicons’.
• Size- 1 to 1000 kilo bp.
• Related to metabolic activity.
• Allows bacteria to reproduce under
unfavorable conditions.
Plasmid
Nomenclature
Lower case P (p)
First letters of researchers name or place
where it was discovered.
Numerical numbers given by workers.
Plasmid
Eg. Plasmid pBR 322
BR is for Bolivar and Rodriguez, who designated it
as 322
Plasmid
Eg. Plasmid pUC 19
UC stands for University of California
Plasmid- Cosmids
• Cosmids are plasmids with cos sequence.
• They are able to accommodate long DNA fragments
that plasmids can’t.
Bacteriphages
• A bacteriophage is a virus that infects
bacteria.
• Virulent portion is deleted.
Genetic material can be
ssRNA, dsRNA, ssDNA,
dsDNA.
Vectors used
• For Single genes- Plasmids are used
• For Large pieces of DNA- Bacteriophages
Phage Lambda () as vector
• 48.5 kb in length.
• Cos sites of 12 bp at the ends.
• Cohesive ends allow circularizing DNA in host.
Lytic Cycle (replication of bacteriophges)
(1) Phage attaches to a specific host
bacterium.
(2) Injects its DNA,
(3) Disrupting the bacterial genome and
killing the bacterium, and
(4) Taking over the bacterial DNA and
protein synthesis machinery to make
phage parts.
(5) The process culminates with the
assembly of new phage, and
(6) The lysis of the bacterial cell wall to
release a hundred new copies of the
input phage into the environment.
Restriction fragments
• A restriction fragment is a DNA fragment
resulting from the cutting of a DNA strand by
the restriction enzyme.
• Process is called restriction.
Restriction fragments
• Steward Linn along with Werner Arber in 1963
isolated two enzymes.
• One of them is Restriction Endonuclease.
• Restriction Endonuclease can cut DNA.
• Restriction Endonuclease are basic
requirement for gene cloning or rDNA
technology.
Restriction fragments
Nucleases
They remove
nucleotides
from the ends
of the DNA
Exonuclease
They make cuts
at specific
Endonucleaspositions within
e
the DNA
Types of REN
REN
Type I
Type II Type III
Mostly used in rDNA technology.
More than 350 types of type II
endonucleases with recognition sites are
known.
Can be used to identify and cleave within
specific DNA.
Nomenclature of ren
• First letter- genus name of bacteria (in italics).
• Next- first two letters of the species name (in
italics).
• Next- strain of the organism.
• Roman number- order of discovery.
Nomenclature of ren
• Eg. - EcoR I
E- Escherichia, co- coli, R-strain Ry 13,
I- first endonuclease to be discovered.
• Eg.- Hind III
H- Haemophilus, in- influenzae, d- strain Rd,
III- third endonuclease to be discovered.
Recognition sequence (restriction
sites)
• It is the site/ sequence where REN cuts the
DNA.
• Sequence of 4-8 nucleotides.
• Most restriction sites are Palindromes.
• In DNA, palindrome is a sequence of base
pairs that reads the same on the two strands
when orientation of reading is kept same.
Cleavage patterns of ren
• REN recognizes the
restriction site.
• Cleave the DNA by
hydrolyzing
Phosphodiester bonds.
• Isolate a particular gene.
• Single stranded ends
called sticky ends.
• These sticky ends can
form hydrogen bonded
base pairs with
complementary sticky
ends or any other
cleaved DNA.
Cleavage patterns of ren
• Restriction fragments
yield a band pattern
characteristic of the
original DNA molecule
& restriction enzyme
used.
Bands
Gel
electrophoresis
Preparing and cloning a DNA library
• Collection of DNA fragments from a particular
species that is stored and propagated in a
population of micro organisms through
molecular cloning.
Genomic library
• Collection of all clones of DNA fragments of
complete genome of an organism.
• All DNA fragments are cloned and stored as
location of desired gene is not known.
• Screening of DNA fragments can be done by
Complementation Or by using Probes.
Construction of Genomic Library.
Entire genome isolated
Cut into fragments by REN
Fragments inserted in Vector
Recombinant vectors are
transferred into suitable organism
Transferred organisms are
cultured and stored
C
dna library
• cDNA is Complementary DNA.
• Produced using Teminism i.e. Reverse
Transcriptase.
• Constructed for eukaryotes.
cDNA is made from mRNA
Start
AAAAAAA
Stop
Mature mRNA
TTTTTTT
Add polyT primer, nucleotides,
and Reverse Transcriptase
AAAAAAA
TTTTTTT
DNA/RNA
RNA removed (by NaOH) and
second strand synthesized
TTTTTTT
Complementary DNA cDNA
Gene Amplification (PCR)
• It is obtaining multiple copies of a known DNA
sequences that contain a gene.
• Done artificially by using PCR (Polymerase
Chain Reaction)
PCR (Polymerase Chain Reaction)
• Developed by
in 1983.
Kary Mullis
• In Vitro technique.
• Scientific technique to generate billions of
copies of a particular DNA sequence in a short
time.
PCR Machine
Requirements for PCR technique
A DNA segment 10035,000 bp in length
to be amplified.
DNA
segment
Primers
Primers-forward and
reverse, are synthetic
oligonucleotides and
complementary to the
desired DNA segment
PCR
Four types of
deoxyribonucleotide
s i.e. dCTP, dGTP,
dTTP, dATP
dNTPs
Thermosta
ble DNA
polymerase
Enzyme that can
withstand upto 94°
C.
Steps of PCR technique
The double strand melts open to single
stranded DNA, all enzymatic reactions stop
(for example : the extension from a previous
cycle).
Ionic bonds are constantly formed and
broken between the single stranded primer
and the single stranded template. Once
there are a few bases built in, the ionic bond
is so strong between the template and the
primer, that it does not break anymore.
The bases (complementary to the template)
are coupled to the primer on the 3' side (the
polymerase adds dNTP's from 5' to 3',
reading the template from 3' to 5' side,
bases are added complementary to the
template)
The exponential amplification of the gene in
PCR.