Chapter 3.1 Flashcards by Jasmine

Applications of recombinant DNA technology: Transgenic animals (5)

Improved farm animals;
Pharming
Disease models
Biopolymer
Xenografting

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Applications of recombinant DNA technology: Transgenic plants (5)

Stress tolerant plants
Improved productivity
Therapeutic proteins
small molecules
Vaccines

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Applications of recombinant DNA technology: Nucleic acids (5)

Gene therapy
Targeted
Diagnostic probes
Vaccines
Anti sense

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Applications of recombinant DNA technology: Recombinant microbes (5)

Industrial enzymes
Vaccines
Therapeutic proteins
Biopolymers
Bioremediation

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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

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genetic engineering often relies on ___ and ___ to modify an organism’s genome

recombinant DNA technology and gene cloning

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Enzymes in recombinant DNA technology (3)

Nucleases
DNA modifiers
DNA ligases

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Nucleases (3)

restriction endonucleases;
Restriction exonucleases;
Ribonuclease H

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DNA Modifiers (6)

DNA polymerase;
Reverse transcriptase;
Alkaline phosphatase
Polynucleotide kinase;
Terminal nucleotidyl transferase;
Methyl transferase

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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

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synthesize complementary
strand (cDNA) from mRNA template

Reverse transcriptase/ RNA dependent
DNA polymerase

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Functions of Reverse transcriptase/ RNA dependent
DNA polymerase (3)

Synthesize cDNA;
Amplify cDNA;
Analyze mRNA

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recognize and cut
DNA strand at specific sequence called restriction
site.

Restriction endonuclease-

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3 types of Restriction endonuclease

Type I RE, Type II RE,
Type III RE

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Type of RE?
-recognize a
bipartite sequence, but do not
produce a predictable cleavage
pattern.

Type 1 RE

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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

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Type of RE?
-recognize a non
palindromic
comprising
oriented site

Type III RE

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subtypes of Type II RE (4)

IIP; IIS; IIC; IIT

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3-protein heterocomplex of Type 1 RE

endonuclease protein (R); methyl transferase protein (M); Specificity protein (S)

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2-protein heterocomplex of Type 3 RE

mod subunit (M)- site recognition & methylation;
res subunit (R)-endonuclease and helicase domains

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are enzymes
composed of distinct domains that exhibit different biochemical activities; converts
blunt end of DNA fragments into sticky end

Terminal transcriptase

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are helpful in cloning because they hold two
pieces of DNA together so they can be linked by
DNAligase

Sticky ends

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2 process of Terminal transcriptase

Reverse transcription process;
RNase H activity of reverse
transcriptase process

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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

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___ 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)