Chp. 9 A Flashcards
What is recombinant DNA?
DNA from two organisms (eg plasmid + foreign DNA)
What is the process of making a recombinant cell?
- Gene of interest is inserted into a self-replicating vector through the use of restriction enzymes which create sticky ends
- Recombinant vector is taken up by a bacterium or a yeast cell
- Cell is allowed to divide many times, producing a culture of clones. All the clones are identical and carry the vector with gene of interest
- Expression of the gene of interest within the clones can produce a large volume of product (protein, enzyme, hormone, etc
How are recombinant cells useful to humans?
- They can be used in therapeutic protein production
- Vaccine development
- Can correct genetic defects in gene therapy
What are some products that are harvested from recombinant cells?
- Proteins
- Enzymes
- Hormones
The process in which organisms with traits better suited to their environments survive and reproduce rather than those that lack those traits is known as ________
Natural selection
The process in which humans actively choose and breed organisms with specific desirable traits is known as ______
Artificial selection
A targeted and specific change in a gene is known as ______.
Why is this important in science?
Site-directed mutagenesis
It is used to study the structure and functions of proteins or DNA, which is valuable for understanding how genetic diseases work.
What are restriction enzymes?
Specifically cut sequences of DNA that recognize specific DNA sequences and cut the DNA at that site
Where do restriction enzymes come from?
They naturally occur in species of bacteria
Why can’t restriction enzymes cut bacterial DNA?
The presence of methylated cytosines does not allow the restriction enzyme to recognize it and therefore will not cut it
A sequence that reads the same forwards and backwards is known as a _____
Palindromic sequence
How does the blunt end and the sticky ends of restriction enzymes work?
The blunt ends are produced when the enzyme cuts the DNA at the same point whereas the sticky ends are produced with overhangs which make it easy for creating recombinant DNA
Why are restriction enzymes important in genetic engineering?
They cut DNA in a predictable and controllable manner
What is a vector?
A piece of DNA that can replicate by itself and is used to transport foreign DNA into a cell (eg. plasmid)
What are the characteristics of a good vector?
- able to replicate autonomously
- small in size
- can be easily isolated
What is the process of increasing small quantities of DNA for analysis called?
Polymerase Chain Reaction (PCR)
What is PCR used for?
Used for diagnostic tests for genetic diseases and detecting pathogens
What are the steps of PCR? (Possible short answer)
- Denaturation @94 deg C breaks hydrogen bonds and separates the DNA strands
- Annealing @55-65 deg C, primers attach to DNA
- Elongation/ extension @72 deg C: taq polymerase adds nucleotides and elongates the DNA
What is reverse transcription and what is it used for?
It is the synthesis of DNA from an RNA template.
Used for providing a means of synthesizing eukaryotic genes from mRNA transcripts
What is reverse transcriptase?
Enzyme that makes a DNA copy of RNA- cDNA
How are genes cloned? (possible short answer)
It is isolated, then inserted into a self-replicating vector. The vector is then introduced into a host cell, which then replicates the vector, which includes the cloned gene
What are the 5 ways of getting DNA into a cell?
- Transformation
- Electroporation
- Microinjection
- Gene gun
- Protoplast fusion
Explain transformation
Cells take up DNA from the surrounding environment
Explain electroporation
Electrical current forms pores in cell membranes
Explain microinjection
Fine glass needle is used to carefully pierce cell membrane and deliver desired substance into cytoplasm or nucleus
Explain the gene gun method
Physically shooting DNA-coated micro particles into the target cells using a device that accelerates these particles
Explain the protoplast fusion method
Removing cell walls from two bacteria which allows them to fuse together
What is the genomic library and what is its purpose?
A collection of clones containing different DNA fragments
They are used as a source of DNA for obtaining eukaryotic genes because eukaryotic DNA has introns that do not code for protein
How is a genetic library made?
An organism’s DNA is digested and spliced into plasmic or phage vectors, and is introduced into the bacteria
What is colony hybridization?
A method that uses DNA probes to select clones
What is cDNA?
DNA made from mRNA by reverse transcriptase
Describe the process of making cDNA (possible essay question)
- gene composed of introns and exons is transcribed to RNA by RNA polymerase
- Processing enzymes in the nucleus remove the intron-derived RNA and splice together the exon-derived RNA into mRNA
- mRNA is isolated from the cell, and reverse transcriptase is added
- First strand of DNA is synthesized
- The mRNA is digested by reverse transcriptase
- DNA polymerase is added to synthesize second strand of DNA
TRUE OR FALSE:
cDNA is necessary when making a recombinant cell with eukaryotic genes because DNA does not have introns, which are needed to code for protein
FALSE
Eukaryotic DNA does have introns, which DO NOT CODE FOR PROTEINS
Short segments of single stranded DNA complementary to the desired gene are known as _______
DNA probes
Describe the clone selection process
:(
What are the desirable traits of a microbial host?
- rapid turnover, fast growth rate
- can be grown in large quantities using ordinary culture methods
- Nonpathogenic
- Well mapped genome
- Can accept plasmid or bacteriophage vectors
- Maintains foreign genes through multiple generation
- Will secrete a high yield of proteins from expressed foreign genes
How are DNA segments sequenced? (Possible essay question)
- Extract genomic DNA containing introns and exons
- Digest DNA and plasmid with same restriction enzyme
- Ligate the digested plasmid and DNA
- Transformation: using CaCl2 and heat shock, the plasmid was inserted in the cell
- Plate the bacteria (each colony has a plasmid with a different fragment of DNA)
- Grow each colony
- Extract plasmid from each colony
- Sequence each plasmid
- Repeat steps 2-8 with multiple restriction enzymes
- Look for overlapping regions
What is the purpose of western blotting?
To use antibodies to detect and identify specific proteins within a mixture of proteins.
