Overview of recombinant DNA technology 6

1. Isolate plasmid 2. Cleave DNA to obtain desired genomes 3. Isolate gene 4. Insert gene into plasmid 5. Insert plasmid (w/ desired gene bacteria) 6. Culture bacteria for various use

Recombinant DNA Technology Flashcards by Sandra Perez

Intentionally modifying genomes of organisms for practical purpose

Recombinant DNA technology

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3 Goals of recombinant DNA technology

Eliminate
Combine
Create

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3 Goals of recombinant DNA technology

-Eliminate

Undesirable Phenotypic traits

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3 Goals of recombinant DNA technology

-Combine

Beneficial traits of two or more organisms

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3 Goals of recombinant DNA technology

-Create

Organisms that synthesize products human need

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Overview of recombinant DNA technology

Isolate plasmid
Cleave DNA to obtain desired genomes
Isolate gene
Insert gene into plasmid
Insert plasmid (w/ desired gene bacteria)
Culture bacteria for various use

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Physical and chemical agents that produce mutations

Mutagens

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Scientists utilize mutagens to

Create changes in microbes’ genomes to change phenotypes

- Select for and culture cells with beneficial characteristics

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____________ alone can be isolated.

Mutated genes

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Explain reverse transcriptase

Isolated from retroviruses (Genome is RNA)
Uses mRNA template to transcribe molecule of cDNA
Easier to isolate mRNA molecule for desired protein first
mRNA of eukaryotes has introns (non-coding regions) removed……. (leaving only coding DNA)
Allows cloning in prokaryotic cells

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Molecules of DNA and RNA produced in cell-free solutions

Synthetic Nucleic Acids

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Uses of synthetic nucleic acids

Elucidating the genetic code

Creating genes for specific proteins

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Identifying codons for a.a

Elucidating the genetic code

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Synthesize a gene for practical use

Creating genes for specific proteins

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Synthesizing DNA and RNA “prodes” to locate specific sequences of nucleotides and Synthesizing antisense nucleic acid molecules use

synthetic nucleic acids con’t

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Prodes are small sequences of nucleic acid which are labeled with fluorescent marker (enables the identification and presence of specific DNA sequences in certain genes)

Synthesizing DNA and RNA “prodes” to locate specific sequences of nucleotides

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Nucleic acid sequence which binds and interferes with genes and/or mRNA to prevent expressions (research conducted to control diseases)

Synthesizing antisense nucleic acid molecules

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Bacterial enzymes that cut DNA molecules only at restriction sites (nucleotide palindromes)

Restriction enzymes

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Categorized into 2 groups based on type of cut

Sticky ends

Blunt ends

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R.E. stands for

Restriction Enzymes

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R.E. are named according to

Genus species
Order found
Strain

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E.Coli strain R has restriction enzyme

ECoR1

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The same____can be used in different organisms

R.E.

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After RE cut DNA, ligase can be added to

Ligate the DNA strand

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Nucleic acid molecules that deliver a gene into a cell

vectors

What are some useful properties of vectors?

They are small enough to manipulate in a lab survive inside cells contain recognizable genetic marker ensure genetic expression of gene

Vectors include

Viral genosomes transposens and plasmid

Growth on antibodic medium indicates that gene

was delivered succuesfully

A collectin of bacterial or phage clones

Gene libraries

Each clone in library often contains

one gene of an organisms genome

Library may contain all genes of a

single chromosomes

librarys may also contain set of

cDNA complementary to mRNA

Production of a gene library

1- isolate genome (all DNA) 2- cleave genome into fragments using RE 3- insert each fragment into a vector (plasmid) 4- insert each vector into different cells 5- culture cells, then place into tubes (label, store)

Copying (amplifying) large number of identical molecule of DNA molecules

The polymerase chain reaction (PCR)

Critical to amplify DNA in variety of situations

Epidemiologists use to amplify genome of unknown pathogen | -amplified DNA from Bacillus anthracis spores in 2001 to identify source of spores

DNA specific, dNTPs polymerase enzyme, thermocycler

PCR requires

Heat DNA `94 C breaks H-bonds between dsDNA, resulting in 2 separate complementary strands

Denaturation

Mixture is cooled to ~65 C, so primers in a mixture can anneal to a complementary sequence in template strands of DNA

Priming

Temperature is slightly elevated to 72 C and polymerase enzyme synthesizes each template strand of DNA using dNTPs

Extension

PCR can be automated using a _______: machine which heats and col PCR mixture. repeats the 3 steps

Thermocycler

PRC stand for

Polymerase chain reaction

PCR

Hydrogen bonds breaks Primers anneal to specific DNA sequences Polymerization occurs, new strands synthesize from ssDNA

PCR DNA is replicated exponentially after

Sever cycles

Gel electrophoresis

Separating DNA Molecules

Explain Gel electrophoresis

Separates molecules based on electrical charge, size, and shape - Allows scentists to isolate DNA of interest - Negatively charged DNA drawn toward positive electrode - Agarose makes up gel; acts as molecular sieve - Smaller fragments migrate caster then larger ones - Determines soze by comparing migrated to strandards

Migration of DNA

smallest DNA fragments travel the fastest

DNA transferred from gel to nitrocellulose membrane

Southern blot

Southern blot probes uses to localize ________

DNA sequence of Interest

what uses southern blots

Genetic "fingerprinting" | Diagnosis of infectious disease

Used to detect RNA

Northen Blot

Explain the southen blot technique (4 Steps)

Cut DNA using RE and place cut DNA mixture into separate wells 1) use gel electrophoresis to separate fragments 2) Transfer (blot) DNA fragmens from gel to nitrocellulose membrane 3) Add radioactive probes to membrane 4) incubate and observe for presence of specific DNA sequence

Goal of DNA technology is insertion of DNA into ____

Cell

Uptake of exogenous DNA

Transformation

Viral transfer of DNA from one microbe --->another

Transduction

Transfer to plasmid to another bacteria

Conjugation

Artifical methods are

Electroporation Protoplast fusion Injection

Gene gun and microinjection

Injection

Electrical current ----> holes so DNA enters

Electroporation

Expose microbes to polyethylene glycol to promote fusion of 2 microbes (Recombinant DNA)

Protoplast fusion

Beads coats w/ DNA and shot into target tissue

Injection - gene gun and microinjection

Locating genes and specific nucleotide sequences on a nucleic acid molecule

Genetic mapping

DNA microarrays used to screen individuals for inherited disease caused by mutatuins

Genetic screening

Genetic screening can also identify _____________.

Pathogens DNA in blood or tissue

Identifying individuals or organisms by their unique DNA sequence

DNA fignerprinting (pharmaceutical and therapeutic applications)

Pharmaceutical and therapeutic application

Amplify DNA (PCR) - Cut DNA (using RE) - -Each individual may have diff. fragment size - Run DNA on gen electrophoresis

Missing or defective gens replace with notal copies

Gene therapy

With gene therapy some patients immune system reacts

Nehatively

Patient specimens can be examined for presence of gene sequences unique to certain pathogens

Medical diagnosis

Animals cells, tissue, or organs introduced into human body

Xenotransplants

(Agricultrual applications) | Recombinant plants and animals altered by addition of genes from other organisms

Production of transgenic organisms

(agricultrual applications) | Gen from salmonella conveys resistance to gyphosate (round up)

Herbicide resistance

Farmers can kill weeds without killing crops | Agricultrual applications

Herbicide resistance

(Agricultrual applications) Scientists have removed gene for salt tolerance and inserted into tomato and canola plants Transgenic plants survive produce fruit and remove salt from soil

salt tolerance

(Agricultrual applications) | crops sprayed with genetically modified bacteria can tolerate mild freezes

Freeze resistance

(Agricultrual applications) (bt toxin) naturally occurring toxin only harmful to insects -organic farmers used to reduce insect damage to crops -gene for Bt toxin inserted into various crop plants

Pest resistance

(Agricultrual applications) | tomatoes allowed to ripen on vine and shelf life increase (gene for enzyme that breaks down pectin suppressed)

Improvements in nutritional value and yield

allows cattle to gain weight more reapidly, have meat with lowe fat contect and produce 10% more milk

BGH

Recombinant DNA Technology Flashcards

(77 cards)