CHAPTER 3 Flashcards
Applications of recombinant DNA in Transgenic animals
- Improved farm animals
- Pharming
- Disease models
- Biopolymer
- Xenografting
Applications of recombinant DNA in Nucleic acids
- Gene therapy
- Targeted
- Diagnostic probes
- Vaccines
- Anti sense
Applications of recombinant DNA in Transgenic plants
- Stress tolerant plants
- Improved productivity
-Therapeutic proteins - Small molecules
- Vaccines
Applications of recombinant DNA in recombinant microbes
- Industrial enzymes
- Vaccines
- Therapeutic proteins
- Biopolymers
- Bioremediation
involves using enzymes and various
laboratory techniques to manipulate and
isolate DNA segments of interest
Recombinant DNA technology
cutting and pasting DNA from different
samples/specimen
Recombinant DNA tech
Enzymes in Recombinant tech
Nucleases
DNA modifiers
DNA Ligases
It joins the DNA fragments with cloning vector
DNA ligase
Also known as molecular glues
Ligases
Three steps of DNA ligation:
- DNA ligase self-adenylates
- Adenyl-group transfers to donor DNA
- Phosphodiester bond forms
synthesize complementary
strand (cDNA) from mRNA template
Reverse transcriptase/ RNA dependent
DNA polymerase
Functions of reverse transcriptase
- Synthesize cDNA
- Amplify cDNA
- Analyze mRNA
recognize and cut
DNA strand at specific sequence called restriction
site.
Restriction Endonuclease
3 types of Restriction endonuclease
Type I
Type II
Type III
-recognize a
bipartite sequence, but do not
produce a predictable cleavage
pattern
Type I RE
are most
commonly used for molecular
biology applications, as they
recognize stereotypical
sequences and produce a
predictable cleavage pattern.
Type II RE
recognize a nonpalindromic sequence,
comprising two inversely
oriented site
Type III RE
are enzymes
composed of distinct domains that exhibit
different biochemical activities.
Terminal Transcriptase
It converts
blunt end of DNA fragments into sticky end.
Terminal transcriptase
are helpful in cloning because they hold two
pieces of DNA together so they can be linked by
DNA ligase.
Sticky ends
Sticky ends have __________ _______ at the end of the
fragment, whereas blunt ends produce _________ __________
Unpaired bases, straight cleavage
It usually cut DNA on either
side of distortion caused by thymine
dimers or intercalating agents.
Nuclease
Two types od nuclease
Endonuclease, Exonuclease
hydrolyzing enzyme that
cleaves the phosphodiester bond between the nucleotides
Endonuclease
Cleave from the ends
Exonuclease
-synthesize nucleotide
complementary to template strand and
helps to fill gap in double stranded DNA.
DNA polymerase
RNase-H removes
mRNA from DNA-RNA heteroduplex and
that mRNA is used to synthesize cDNA
Ribonuclease-H
helps in
removal of terminal phosphate group
from 5′ end.
Alkaline phosphatase
It adds
phosphate group from ATP molecule to
terminal 5’end after dephosphorylation
by alkaline phosphatase
Polynucleotide kinase
enzymes that
transfer a methyl group from Sadenosylmethionine (SAM) to their substrates.
Methyltransferases
DNA methyltransferases modify DNA by
adding a methyl group to _____________.
Cytosines
derived from a Greek word that describes
a cutting (of a twig) that used to propagate
or copy a plant.
Clone
a molecule, cell, or organism produced
from another single entity to which it shares
the same genetic make-up.
Clone
the process of producing or
generating a genetically identical
copy of a cell or an organism.
Cloning
DNA cutting enzymes, also known as
“scissors” used for gene cloning
Restriction Enzymes
restriction endonucleases (endo,
“__________”, nuclease, “_____________________”)
Within, nucleic acid-cutting enzyme
they cut within DNA sequences as
opposed to enzymes that cut from the ends of
DNA sequences (exonucleases)
Restriction endonucleases
Are the DNA molecules, which can carry a foreign DNA fragment into a host cell
Vectors
If it is used for the reproducing the DNA fragment, called _______ _______
Cloning vector
Vector is also called _______ _____ because they act as carrier of gene to be cloned into a recipient cell.
vehicle DNA
Properties of vector
- Small DNA molecule
- Origin of replication
- Unique restriction enzyme are able to replicate autonomously
- Be non-toxic to cell
- Have space for foreign insert
- Have suitable marker genes
- Unique recognition sites
are DNA molecules
into which foreign DNA can be
inserted
Cloning vectors
_________ is an artificially synthesized/
manipulated DNA whereas a _________
naturally occurs in bacterial cells.
Vector, Plasmid
There are several vectors, which can be
used in recombinant DNA, whereas all
__________ may not be used directly in
recombinant DNA technology.
Plasmids
Components of an empty plasmid vector
Promoter, Restriction site, Origin of replication, Antibiotic resistance gene
Components of a recombinant plasmid vector
Promoter, Restriction sites, Inserted DNA fragment, Origin of replication, Antibiotic resistance gne
Preparation of recombinant plasmid vector
- Cleave vector with restriction enzyme
- Cleave foreign DNA with same restriction enzyme
- Mix vector and DNA fragment under conditions that favor base pairing
- Treat with DNA ligase to join DNA pieces covalently
or origin of replication, is the sequence
at which replication of DNA begins.
Ori
There are only ____ selectable markers
two
are genes that help identify
bacteria that have successfully transformed, or
taken up the recombinant plasmid.
Selectable markers
are commonly used markers.
Antibiotic resistance genes
It acts as a light switch, and it signals when to turn the gene on and off
Promoter
r is a short region of DNA (100–1,000
bp) where transcription of a gene by RNA
polymerase begins. It is typically located directly
upstream or at the 5′ end of the transcription
initiation site.
Promoter
Characteristics of cloning vectors
- Self-replicating inside host cell
- Unique restriction enzyme
- possess some marker gene
- easily isolated from the host cell
- Unaffected by the introduction of donor DNA
fragment in terms of its replication property
Types of cloning vectors
- Bacteriophage
- Plasmid DNA/Plasmid vetcor
- Bacterial Artificial Chromosomes (BACs)
- Yeast Artificial Chromosomes (YACs)
- Human Artificial Chromosomes (HACs)
these are viruses, known as
phage, which can infect bacterial
cells
Bacteriophage
capable to deliver DNA fragment
of a size up to 20 kb
Bacteriophage
Parts of Bacteriophage:
Spikes, Long tail fibre, Baseplate, Tail tube and sheath, Collar, Nucleic acid (DNA), Capsid
small circular pieces of DNA found in primarily in
bacteria
Plasmid DNA/ Plasmid Vector
considered as extrachromosomal DNA in bacteria
(found in the cytoplasm in addition to the bacterial
chromosome)
Plasmid DNA
plasmids may be inserted into bacterial cells in
the process known as
Transformation
a DNA fragment of size up to 10 kb can be
delivered using this vector
Plasmid DNA
plasmid which is designed to clone
very large DNA fragments ranging in
size from 75 to 300 kb
Bacterial Artificial Chromosomes (BACs)
used in sequencing the genome of
organisms in genome projects i.e.,
human genome project
BACs
yeast expression vectors
- may clone DNA fragments with sizes from 100
kb to 3000 kb
Yeast Artificial Chromosomes (YACs)
used for cloning very large DNA fragments and
for the physical mapping of complex genomes
YACs
However, YACs are known to produce _________
effects which make them less stable compared
to BACs
Chimeric
- An organism or tissue that contains at least two different
sets of DNA, most often originating from the fusion of as many different
zygotes (fertilized eggs).
Chimerism
artifacts where the
sequence of the cloned DNA actually
corresponds not to a single genomic region
but to multiple regions.
Chimeric effects
o a DNA fragment that
consists of DNA from two or more different
sources
Chimeric DNA
Developed by Burke and Olson in 1987
YAC vectors
Can replicated inside yeast; used to clone DNA sequences in yeast cells
YAC vectors
Developed by Melsimon et al in 1992; can replicate inside bacteria
BAC
also known as mammalian artificial
chromosomes (MACs)
-still under development
Human Artificial Chromosomes (HACs)
range in size from 6 to 10 Mb that carry
new genes introduced by human researchers
HACs
- used as vectors in transfer of new genes,
studying their expression, and mammalian
chromosomal function can also be elucidated
using these micro chromosomes in
mammalian system.
HACs
DNA fragment/s from
two different species
that are inserted into a
host organisms to
produce new genetic
combinations that could
add value to science,
agriculture, medicine,
and industry.
Recombinant DNA
many fragments are randomly cloned at
once and no individual gene is specifically
targeted for cloning.
Shotgun cloning
Creating recombinant DNA:
- Isolate DNA from two sources
- Cut both DNAs with the same restriction enzyme
- Mix the DNAs; they join by base pairing
- Add DNA ligase to seal DNA with covalent bonds
- Put plasmid into bacterium by transformation
- Clone the bacterium
a method for the identification and cloning of
genes which includes a DNA library
Cloning approach involving DNA libraries
collection of cloned
DNA fragments from a particular
organism contained within bacteria or
virus as a host
DNA library
is synthetic DNA
that has been transcribed from a specific mRNA
through a reaction using the enzyme reverse
transcriptase
Complementary DNA
the chromosomal DNA (which the whole
genome of the organism) from the tissue of
interest is isolated and then digested with
restriction enzyme .
Genetic library
mRNA from the tissue of interest is
isolated and used for making the library
cDNA library
after building
the genomic library or cDNA library, it must
be screened to identify the genes of
interest
- colony hybridization
Library Screening
a more rapid approach to cloning
compared to building and screening a
library - a technique for making copies or
amplifying a specific sequence of DNA in
a short period of time.
Polymerase Chain Reaction (PCR)
Some Laboratory Techniques and Application
of Recombinant DNA Technology
Agarose gel electrophoresis, DNA Sequencing, Next generation sequencing, Fluorescence in situ hybridization (FISH), Southern blotting, Northern blot analysis
allows one to separate and visualize DNA
fragments based on size
Agarose gel electrophoresis
- gel with a high percentage of agarose (say 2%)
is better suited for separating ______DNA
fragments while lower percentage of agarose is
better suited for resolving ______ DNA fragments.
small, large
a technique of
determining the nucleotide sequence of the
gene – the exact order of the bases in the
genome or gene of the organism
DNA sequencing
designed to produce highly accurate and
long stretches of DNA sequence, greater than
1 giga base (billion bases) of DNA per reaction,
at a low cost
Next Generation Sequencing
- can be used to identify which chromosome
contains a gene of interest - can also be used to determine the cell type that
is expressing a particular mRNA
Fluorescence In Situ Hybridization
Process of Next Generation Sequencing
- DNA extraction
- Library preparation
- Sequencing
- Analysis
molecular biology technique for identification
and quantification of DNA
Southern blotting
- following electrophoresis, the gel is treated
with an alkaline solution to denature the DNA;
then the fragments are transferred onto a
nylon or nitrocellulose membrane using a
technique called blotting - used to visualize only specific fragments
of interest
Southern blotting
Process of Southern blotting
- Extract DNA from the sample
- Fragment DNA with restriction enzymes
- Separate DNA by size using gel electrophoresis
- Transfer and fix separated DNA molecules from the gel into a nylon membrane
- Probe for the DNA of interest using a nucleotide probe
- Radiolabeled probes can be analyzed
- molecular biology
technique for identification and
quantification of DNA
Northern blotting
RNA is isolated from a tissue of interest and
separated by gel electrophoresis (the RNA is
not digested with enzymes)
Northern blotting
RNA is blotted onto a nylon membrane and
then hybridized to a probe - Amounts of mRNA
produced by different tissues can be compared
and quantified via different techniques
Northern blotting
performed when only very small amount of
mRNA is extracted from the sample; isolated
mRNA is converted into double-stranded
cDNA by the enzyme reverse transcriptase in a
process similar to the way in which cDNA for a
library is made.
Reverse transcription PCR (RT-PCR)
enables researchers to quantify amplification
reactions as they occur in “real time”; basic
procedure involves the use of specialized
thermal cyclers that use a laser to scan a beam
of light through the top or bottom of each PCR
tube
real-time PCR or quantitative PCR
– another technique for studying gene
expression; created with the use of a small
glass microscope slide; single-stranded DNA
molecules are attached or “spotted” onto the
slide using a computer-controlled highspeed robotic arm called an arrayer, which
is fitted with a number of tiny pins; arrayer
fixes the DNA onto the slide at specific
locations
Gene microarrays/DNA microarrays/gene chip
mutations can be
created in specific nucleotides of a cloned gene
contained in a vector; gene can then be
expressed in cells, which results in the translation
of a mutated protein; site-directed mutagenesis
can be a very valuable way to help scientists
identify critical sequences in genes that
produce proteins involved in human Diseases.
Gene mutagenesis studies
a technique that uses
double-stranded pieces of RNA (dsRNA) to
inhibit or silence expression of genes
RNA interference
