1. Restriction endonucleases 1. restriction exonucleases 1. ribonucleases H

3: Recombinant DNA Technology Flashcards by porenz m

4 Applications of Recombinant DNA Technology

Transgenic animals
Nucleic Acids
Transgenic plants
Recombinant Microbes

How well did you know this?

Not at all

Perfectly

Recombinant DNA technology involves using _ and _ to manipulate and isolate DNA segments of interest.

enzymes
various laboratory techniques

How well did you know this?

Not at all

Perfectly

cutting and pasting DNA from different
samples/specimen

Recombinant DNA Technology

How well did you know this?

Not at all

Perfectly

_ often relies on recombinant DNA technology and gene cloning to modify an organism’s genome

genetic engineering

How well did you know this?

Not at all

Perfectly

3 Classifications of Enzymes in Recombinant DNA Technology

Nucleases
DNA modifiers
DNA ligase

How well did you know this?

Not at all

Perfectly

3 Nucleases

Restriction endonucleases
restriction exonucleases
ribonucleases H

How well did you know this?

Not at all

Perfectly

6 DNA modifiers enzyme

DNA polymerase
Reverse transcriptase
alkaline phosphatase
polynucleotide kinase
terminal nucleotidyl transferse
methyl transferase

How well did you know this?

Not at all

Perfectly

enzymes that joins the DNA fragments with
cloning vector

DNA ligase

How well did you know this?

Not at all

Perfectly

3 Steps of DNA Ligation

DNA ligase is self-adenylated
Adenyl group is transferred
Phospodiester bond forms

How well did you know this?

Not at all

Perfectly

enzyme that synthesize complementary
strand (cDNA) from mRNA template

reverse transcriptase/
RNA dependent DNA polymerase

How well did you know this?

Not at all

Perfectly

3 Functions of reverse transcriptase/
RNA dependent DNA polymerase

synthesize cDNA
amplify cDNA
analyze mRNA

How well did you know this?

Not at all

Perfectly

enzyme that recognize and cut DNA strand at specific sequence called restriction site

restriction endonuclease

How well did you know this?

Not at all

Perfectly

3 types of restriction endonucleases

Type 1 RE
Type II RE
Type III RE

How well did you know this?

Not at all

Perfectly

3 types of restriction endonucleases

recognize a bipartite sequence but do not produce a predictable cleavage pattern

Type 1 RE

1 enzyme w 3 subunits for recognition cleavage and methylation

1000bp from recognition site
E. coli
Citrobacter fruendii

How well did you know this?

Not at all

Perfectly

3 types of restriction endonucleases

most commonly used for molecular biology applications as they reconize stereotypical sequences and produce a predictable cleavage pattern

Type II RE

2 diff enzymes; same as recognition/ close to recognition site

Escherichia coli
Arthrobacter luteus

How well did you know this?

Not at all

Perfectly

3 types of restriction endonucleases

recognize a non-palindromic sequence, comprising two inversely oriented sites

Type III RE

1 enzyme with 2 subunits; 24-26 bp from recognition site

Haemophilus influenzae
Providencia stuarti

How well did you know this?

Not at all

Perfectly

Enzymes used in recombinant DNA technology

are enzymes composed of distinct domains that exhibit different biochemical activities.

It converts blunt end of DNA fragments into sticky end.

terminal transcriptase

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

How well did you know this?

Not at all

Perfectly

Enzymes used in recombinant DNA technology

It usually cut DNA on either side of distortion caused by thymine dimers or intercalating agents.

Nuclease

How well did you know this?

Not at all

Perfectly

Enzymes used in recombinant DNA technology

synthesize nucleotide complementary to template strand and helps to fill gap in double stranded DNA.

DNA polymerase

How well did you know this?

Not at all

Perfectly

Enzymes used in recombinant DNA technology

removes mRNA from DNA-RNA heteroduplex and that mRNA is used to synthesize cDNA

Ribonuclease-H

How well did you know this?

Not at all

Perfectly

Enzymes used in recombinant DNA technology

helps in
removal of terminal phosphate group
from 5′ end

alkaline phosphatase

How well did you know this?

Not at all

Perfectly

Enzymes used in recombinant DNA technology

It adds phosphate group from ATP molecule to
terminal 5’end after dephosphorylation
by alkaline phosphatase

Polynucleotide kinase

How well did you know this?

Not at all

Perfectly

Enzymes used in recombinant DNA technology

enzymes that transfer a methyl group from S-denosylmethionine (SAM) to their substrates.
DNA methyltransferases modify DNA by
adding a methyl group to cytosines.

Methyltransferases

How well did you know this?

Not at all

Perfectly

Type of Nuclease

hydrolyzing enzyme that
cleaves the phosphodiester bond between the nucleotides

endonuclease

How well did you know this?

Not at all

Perfectly

# Type of Nuclease cleave from the ends

exonuclease

Clone is derived from Greek word that describes _ that used to propagate or copy a plant

cutting (of a twig)

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 enzymes aka _ because 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 self replicating

vectors

it is used for the reproducing the DNA fragment

cloning vector

vectors aka _ because they act as carrier of gene to be cloned into a recipient cell

vehicle DNA

properties of vector

1. **small DNA** molecules 1. **ori**gin of replication 1. **unique restriction enzyme** are able to replicate autonomously 1. be **non-toxic** to host cell 1. have **space** for foreign insert 1. the vector should have suitable **marker genes** that allow easy selection of the transformed host cell 1. **unique recognition site**

are DNA molecules into which foreign DNA can be inserted

cloning vectors

vectors are also known as _ for delivering foreign DNA into recipient cells

vehicles

during _ , the desired DNA is carried by a vector into another cell where it can be replicated and/or expressed

DNA cloning

vector is an artifically synthesized manipulated DNA whereas a _ naturally occurs in bacterial cells

plasmid

there are several _ which can be used in recombinant DNA, whereas all _ may not be used directly in recombinant DNA technology

vectors plasmids

Preparation of recombinant plasmid vector

1. cleave vector with restriction enzyme 1. cleave foreign DNA with same restriction enzyme 1. mix vector and DNA fragment under conditions that favor base pairing 1. treat with DNA ligase to join DNA pieces covalently

the sequence at which replication of DNA begins

ori origin of replication

Ori is also linked to the copy _ and so controls how many times the gene of interest will be produced in the host cell

number of vector

_ are genes that help identify bacteria that have successfully transformed, or taken up the recombinant plasmid _ are commonly used markers

selectable markers antibiotic resistance genes ## Footnote ideally, the enzyme should have 1 or max of 2 recognition sites

why should the number of enzymes be so low

cutting the vector within the selectable marker **will interrupt** that genes, and allow for identification of recombinants ## Footnote ex. using cloning site within an ampicillin resistance gene will result in recombinants that are no longer resistance to that antibiotic

_ region of the gene acts as a light switch signals when to turn the gene on and off

promoter

a short region of DNA (100-1000 bp) where transcription of a gene by RNA polymerase begins

promoter

promoter is typically located directly_ or at the _ end of the transcription initiation site

upstream 5'

# Characteristics of cloning vectors 1. _ inside host cell 1. possess a _ for RE enzymes 1. _ by the introduction of donor DNA fragment in terms of its replication property 1. possess some _ such that it can be used for identification of recombinant cell (usually an antibiotic resistance gene that is absent in the host cell) 1. _ from host cell

1. self-replicating 1. unique restriction site 1. unaffected 1. marker gene 1. early isolated

Types of Cloning vectors

1. bacteriophage 1. plasmid DNA/ plasmid vector 1. bacterial artificial chromosomes BACs 1. yeast artificial chromosomes YACs 1. human artificial chromosomes HACs

# Types of Cloning Vectors are viruses which can infect bacterial cells capable to deliver DNA fragment of a size up to 20kb

bacteriophage

# Types of Cloning Vectors small circular pieces of DNA found in primarily in bacteria considered as extrachromosomal DNA in 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 ## Footnote a DNA fragment of size up to 10kb can be delivered using this vector

transformation

# Types of Cloning Vectors plasmid which is designed to clone very large DNA fragments ranging in size from 75 to 300 kb

Bacterial Artificial Chromosomes (BACs)

Bacterial Artificial Chromosomes (BACs) used in sequencing the genome of organisms in genome projects i.e., _

human genome project

# Types of Cloning Vectors - _ expression vectors - may clone DNA fragments with sizes from 100 kb to 3000 kb - used for cloning very large DNA fragments and for the physical mapping of complex genomes - _ have an advantage over BACs in expressing eukaryotic proteins that require post translational modifications

Yeast Artificial Chromosomes (YACs)

YACs are known to produce _ which make them less stable compared to BACs

chimeric effects

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

"**chimeric effects**" : artifacts where the sequence of the cloned DNA actually corresponds not to a single genomic region but to _

multiple regions. ## Footnote Chimeric DNA refers to a DNA fragment that consists of DNA from two or more different sources

# Type of Cloning Vector * also known as mammalian artificial chromosomes (MACs) * still under development * micro-chromosomes that can act as a new chromosome in a population of human cells * range in size from 6 to 10 Mb that carry new genes introduced by human researchers

Human Artificial Chromosomes (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

Human Artificial Chromosomes (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

2 process of Identifying and Cloning the Gene of Interest

1. Shotgun cloning/Shotgun method/Shotgun approach 1. Cloning approach involving DNA libraries 2.1 DNA Library 2.1.1 Complementary DNA 2.1.2 Genomic Library 2.2 Library Screening 2.3 Polymerase Chain Reaction

# Identifying and Cloning the Gene of Interest many fragments are randomly cloned at once and no individual gene is specifically targeted for cloning.

Shotgun cloning/Shotgun method/Shotgun approach

# Identifying and Cloning the Gene of Interest a method for the identification and cloning of genes which includes a DNA library

Cloning approach involving DNA libraries

# Laboratory Techniques & Application of Recombinant DNA Technology 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 (NGS)

# Laboratory Techniques & Application of Recombinant DNA Technology - 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 (FISH)

# Laboratory Techniques & Application of Recombinant DNA Technology - molecular biology technique for identification and quantification of **DNA** - begins by digesting chromosomal DNA into small fragments with restriction enzymes - 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/Southern blot analysis/Southern hybridization

# Laboratory Techniques & Application of Recombinant DNA Technology - molecular biology technique for **identification and quantification of RNA** - RNA is isolated from a tissue of interest and separated by gel electrophoresis (the RNA is not digested with enzymes) - 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 Blot Analysis

collection of cloned DNA fragments from a particular organism contained within bacteria or virus as a host.

DNA Library

types of libraries for cloning

genomic DNA libraries and complementary DNA libraries (cDNA libraries)

is synthetic DNA that has been transcribed from a specific mRNA through a reaction using the enzyme reverse transcriptase.

complementary DNA (cDNA)

what library talks about the chromosomal DNA (which the whole genome of the organism) from the tissue of interest is isolated and then digested with restriction enzyme .

Genomic Library

Disadvantage of Genomic Library

(1) non-protein-coding pieces of DNA, called **introns, are cloned** in addition to protein-coding sequences (exons); and (2) **eukaryotes have large genomes**

what library talks about the mRNA from the tissue of interest is isolated and used for making the library

cDNA library

mRNA cannot be cut directly with restriction enzymes so it has to be converted to a _

double stranded DNA molecule (complementary DNA or cDNA)

– after building the genomic library or cDNA library, it must undergo _ 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

1. Agarose gel electrophoresis 1. DNA sequencing 1. Next Generation Sequencing (NGS) 1. Fluorescence In Situ Hybridization (FISH) 1. Southern Blotting/Southern blot analysis/Southern hybridization 1. Northern Blot Analysis

# Laboratory Techniques & Application of Recombinant DNA Technology - allows one to separate and visualize DNA fragments based on size - applications generally involve gels that contain 0.5% to 2% agarose

Agarose gel electrophoresis

# Laboratory Techniques & Application of Recombinant DNA Technology 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

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

Benefits of knowing the exact sequence of the gene

1. to **deduce the amino acid sequence** of a protein encoded by a cloned gene 1. to **determine the exact structure of gen**e 1. to **identify regulatory elements** such as promoter sequences 1. to **identify differences in genes** created by gene splicing 1. to **identify genetic mutations**

**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 (qPCR)

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 high-speed 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

3: Recombinant DNA Technology Flashcards

(87 cards)