Recombinant DNA Flashcards
What is genetic engineering?
The deliberate modification of a genomes genetic information by directly changing its nucleic acid genome
What is the definition of a Gene
- A stretch of DNA that codes for a protein which has a specific function
- unit of heredity in organism
DNA does not make the organism, it only makes protein
What are plasmids in genetic engeneering also known for?
Vectors
What is a bacteriophage?
virus that use bacteria cells as host
What is Recombinant DNA Molecules and what is another word for it?
Plasmids that have foreign DNA inserted
Another word is Chimeric Molecules
what is a Clone
an exact copy of the parent
What is Recombinant DNA
When DNA from 2 different organisms are combined to form a new stable fragment of DNA
Explain how scientists can obtain a specific gene sequence through recombinant DNA (rDNA) technology.
- If a specific sequence is desired, it can be added to DNA of another host by rDNA Technology
- specific sequence must be identified and then cut from the DNA strand using Restriction enzymes
- that specific DNA sequence is carried by a vector which transfers the recipient DNA into host DNA
- Ligase then joins the cut DNA in the vector
- vector with DNA sequence is introduced into recipient cell
explain basic steps involved in rDNA or genetic engineering
- construction of recombinant DNA molecule using vector and fragment of DNA that was cut by restriction enzymes
- Transport into host cell
- multiplication of rDNA molecule (in host cell)
- division of host cell
- numerous cell divisions resulting in clone
cells with genes of interest are cloned with either of 2 goals in mind
- create and harvest copies of a gene or
- Ex: gene encoding protein for pest resistance inserted into plant cells
- Ex: gene encoding degradative enzyme to clean up toxic waste is inserted into bacterial cells
- create and harvest protein products of a gene
- Ex: amylase, cellulase, & other enzymes prepares fabrics for clothing manufacture
- Ex: human growth hormone treats stunted growth
what is Reverse transcriptase used for
- used to synthesize a cDNA gene from mRNA template
- makes DNA polymer of an RNA polymer
what are restriction enzymes?
cut DNA molecules only at restriction sites. also called molecular scissors
what are synthetic nucleic acids
Nucleic acid synthesis machines that synthesize molecules over 100 nucleotides long in a few hours
what are Vectors
autonomously replicating genetic element used to carry a fragment of target DNA into host cell for purpose of cloning and expression
what are the tools used in rDNA techonology
- can be physical agents, naturally occurring enzymes and synthetic molecules used to manipulate genes and genomes
- mutagens
- reverse transcriptase
- synthetic nucleic acids
- restriction enzymes
- vectors
list the enzymes that are used in rDNA technology (6).
- restriction endonuclease
- reverse transcriptase
- DNA polymerase
- Polynucleotides
- Alkaline Phosphate
- DNA Ligase
what is a Polynucleotides Kinase?
Adds a phosphate to the 5’-OH end of a polynucleotide, to label it or permit litigation
what is Alkaline Phosphatase?
- a type of enzyme that is used in rDNA technology
- removes terminal phosphate from 5’ or 3’ end of both.
- end modification enzyme by making changes to ends of DNA molecules
what are the recognition sites of Restriction Enzymes? Provide an example.
- Palindrome sequences
- EcoRI has recognition site of GAATTC
what are the 2 possible end results when you break double-stranded DNA using restriction enzymes?
- Sticky end; if one strand extends beyond the complementary region
- Blunt end
Provide example of the 5 common restriction enzymes used and their recognition sites
- EcoRI: 5’GAATTC3’ (sticky)
- BamHI: 5’ GGATCC3’(sticky)
- HindIII: 5’AAGCTT3’(sticky)
- Taq I: 5’ TCGA3’(sticky)
- ECORV: 5’GATATC3’(blunt)
explain the method used to isolate a gene
- lysis method is used, centrifuge and homogenization
- Lysis buffer (basically a detergent) that is used to break the cell wall
- enzymes break down lipid molecules in cell membrane and nuclear membrane
- proteases break down proteins, inactivate macromolecules and RNAases break down RNA
- centrifuge to separate DNA from waste,
- ethanol precipitates DNA
what is PCR and what is it used for?
- Polymerase Chain Reaction
- used to amplify small segments of DNA
what are the components of PCR?
- DNA template
- DNA polymerase
- primers
explain the 3 steps in PCR
- Denaturation: DNA strand heated to 93-95 C in order to break H bonds that hold together 2 strands of DNA
- Annealing: temperature is lowered to allow primers to connect to DNA strand. the temperature depends on primers used (usually 5 C lower then primer melting point)
-
Elongation: DNA polymerase synthesizes complementary strand of DNA, begins from the primer
- DNA polymerase uses dNTP (deoxyribonucleoside triphosphate)
all 3 steps repeat 30-40x which yields billions of target DNA strand
what is Southern Blot used for?
a molecular technique to find target DNA sequence in a sample
what is electrophoresis?
the separation of molecules based on their electrical charge, size and shape
What is Microarray Analysis?
involves breaking open a cell, isolating its genetic components, IDing all cells that are turned on in that particular cell, and making a list of those genes
Microarrays are used in a number of ways, list them.
- monitoring gene expression
- diagnosing infection
- ID organisms in an environmental sample
What are the good characteristics of a vector? (5)
- should be able to self replicate
- have promoter region
- have selectable marker
- have antibiotic resistant gene
- possess a clone site
What are some artificial methods to introduce DNA into cells, explain. (3)
- Electroporation: electrical current applied to cell which makes it competent to take up DAN
- protoplast fusion: enzyme digest cell walls to create protoplast that fuse at a high rate when treated with polyethylene glycol
- microinjection
what are the benefits of synthetic nucleic acids?
- elucidating genetic code
- creating genes for specific proteins
- synthesizing DNA and RNA probes to locate specific sequence of nucleotides
- synthesizing antisense nucleic acid molecules
- synthesizing PCR primers
