Chapter 3.1 Flashcards
Applications of recombinant DNA technology: Transgenic animals (5)
Improved farm animals;
Pharming
Disease models
Biopolymer
Xenografting
Applications of recombinant DNA technology: Transgenic plants (5)
Stress tolerant plants
Improved productivity
Therapeutic proteins
small molecules
Vaccines
Applications of recombinant DNA technology: Nucleic acids (5)
Gene therapy
Targeted
Diagnostic probes
Vaccines
Anti sense
Applications of recombinant DNA technology: Recombinant microbes (5)
Industrial enzymes
Vaccines
Therapeutic proteins
Biopolymers
Bioremediation
involves using enzymes and various laboratory techniques to manipulate and isolate DNA segments of interest;
- cutting and pasting DNA from different samples/specimen
Recombinant DNA Technology
genetic engineering often relies on ___ and ___ to modify an organism’s genome
recombinant DNA technology and gene cloning
Enzymes in recombinant DNA technology (3)
Nucleases
DNA modifiers
DNA ligases
Nucleases (3)
restriction endonucleases;
Restriction exonucleases;
Ribonuclease H
DNA Modifiers (6)
DNA polymerase;
Reverse transcriptase;
Alkaline phosphatase
Polynucleotide kinase;
Terminal nucleotidyl transferase;
Methyl transferase
three steps of a DNA ligation
reaction
- DNA ligase is self-adenylated
- adenyl group is transferred
to 5’ phosphorylated end - Phosphodiester bond forms between the hydroxyl group and the DNA adenylate
synthesize complementary
strand (cDNA) from mRNA template
Reverse transcriptase/ RNA dependent
DNA polymerase
Functions of Reverse transcriptase/ RNA dependent
DNA polymerase (3)
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 RE, Type II RE,
Type III RE
Type of RE?
-recognize a
bipartite sequence, but do not
produce a predictable cleavage
pattern.
Type 1 RE
Type of RE
- are most
commonly used for molecular
biology applications, as they
recognize
stereotypical
sequences and produce a
predictable cleavage pattern.
Type II RE
Type of RE?
-recognize a non
palindromic
comprising
oriented site
Type III RE
subtypes of Type II RE (4)
IIP; IIS; IIC; IIT
3-protein heterocomplex of Type 1 RE
endonuclease protein (R); methyl transferase protein (M); Specificity protein (S)
2-protein heterocomplex of Type 3 RE
mod subunit (M)- site recognition & methylation;
res subunit (R)-endonuclease and helicase domains
- are enzymes
composed of distinct domains that exhibit different biochemical activities; 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
DNAligase
Sticky ends
2 process of Terminal transcriptase
Reverse transcription process;
RNase H activity of reverse
transcriptase process
steps of Reverse transcription process of terminal transcriptase (5)
1.(w/ annealed primer) reverse transcriptase binds to an RNA template and initiates the reaction
2. synthesis of the complementary DNA (cDNA)strand, incorporating dNTPs
3. degrading the RNA
template of the DNA
4. synthesis of second-strand cDNA
5. Formed double-strand cDNA
___ ends have unpaired bases at the end of the
fragment, whereas ___ends produce straight cleavage
Sticky;
blunt
-It usually cut DNA on either
side of distortion caused by thymine dimers or intercalating agents.
Nuclease
2 types of nuclease:
___=hydrolyzing enzyme that
cleaves the phosphodiester bond between the nucleotides
___= cleave from the ends
endonuclease;
exonuclease
-synthesize nucleotide
complementary to template strand and
helps to fill gap in double stranded DNA
DNA polymerase
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 S-adenosylmethionine (SAM) to their substrates
Methyltransferases
__ modify DNA by
adding a methyl group to cytosines.
methyltransferases
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
are DNA molecules which can carry a foreign DNA fragment into a host cell
vector
if it is used for the reproducing the DNA fragment is it called ___
cloning vector
vectors are also called ___ because they act as carrier of gene to be cloned into a recipient cell
vehicle DNA
properties of vectors (7)
-small DNA molecules
-origin of replication
-unique RE replicate autonomously
-non toxic to host cell
- have space for foreign insert
-have suitable marker genes
-unique recognition site
are DNA molecules
into which foreign DNA can be
inserted
* Vehicles for delivering foreign DNA into
recipient cells
Cloning vectors
2 differences between vectors and plasmids:
vector- artificially synthesized/
manipulated DNA; used
in recombinant DNA
plasmid- naturally
occurs in bacterial cells;not be
used directly in
recombinant
DNAtechnology.
, is the sequence
at which replication of DNA begins.
ori, or origin of replication
This sequence is also linked to the copy number of
the vector, and so controls how many times
your gene of interest will be produced in the
host cell
ori, or origin of replication
____are commonly used markers.
Antibiotic resistance
genes
the ___region of the gene acts as a light switch. It signals when to turn the gene on and off
promoter
A ____is a short region of DNA (___ 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; 100–1,000 bp
Characteristics of cloning vectors (5)
-Self-replicating in host cell
- unique restriction site for RE
- Unaffected by the introduction of donor DNA fragments
- possess marker genes
- easily isolated from host
Types of Cloning Vectors (5)
Bacteriophage
Plasmid DNA/plasmid vector
Bacterial Artificial Chromosomes
(BACs)
Yeast Artificial Chromosomes (YACs)
HumanArtificial Chromosomes (HACs)
are viruses, known as
phage, which can infect bacterial
cells-capable to deliver DNA fragment
of a size up to ___ kb
bacteriophage; 20kb
small circular pieces of DNA found in primarily in
bacteria- considered as extrachromosomal DNAin bacteria
(found in the cytoplasm in addition to the bacterial
chromosome
Plasmid DNA/plasmid vector
plasmids may be inserted into bacterial cells in
the process known as ___; a DNA fragment of size up to ___ can be
delivered using this vecto
transformation; 10 kb
plasmid which is designed to clone
very large DNA fragments ranging in
size from ___kb; used in sequencing the genome of
organisms in genome projects
Bacterial Artificial Chromosomes
(BACs); 75 to 300 kb
may clone DNA fragments with sizes from ____- used for cloning very large DNA fragments and
for the physical mapping of complex genomes
Yeast Artificial Chromosomes (YACs); 100 kb to 3000 kb
have an advantage in
expressing eukaryotic proteins that require
post translational modifications; are known to produce chimeric
effects which make them less stable
YACs
___-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
refers to a DNA fragment that
consists of DNA from two or more different
sources.
Chimeric DNA
who developed the:
YACs=
BACs=
YACs= Burke and Olson 1987
BACs= Melsimon et al 1992
micro-chromosomes that can act as a new
chromosome in a population of human cells;
range in size from __ mb that carry new genes introduced by human researchers
HACs or MACs;6 to 10 Mb
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/MACs
DNA fragment/s from
two different species
that are inserted into a
host
organisms
to
produce new genetic
combinations
Recombinant DNA
Identifying and Cloning the Gene of Interest (2)
Shotgun approach;
Cloning approach involving DNA libraries
many fragments are randomly cloned at
once and no individual gene is specifically
targeted for cloning.
Shotgun approach;
a method for the identification and cloning of
genes which includes a DNA library
Cloning approach involving DNA libraries
2 types of shotgun approach
Hierarchical shotgun approach
whole-genome shotgun approach
- collection of cloned
DNA fragments from a particular
organism contained within bacteria or
virus as a host.
DNA Library
types of libraries for cloning (2)
genomic DNA libraries and complementary DNA libraries
___ is synthetic
DNA that has been transcribed from a specific mRNA
through a reaction using the enzyme reverse
transcriptase.
complementary DNA (cDNA)
chromosomal DNA (which the whole
genome of the organism) from the tissue of
interest is isolated and then digested with
restriction enzyme
Genomic Library
non-protein-coding pieces of DNA=
protein-coding sequences=
introns;
exons
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)
