Unit 3 Chapter 19: Bio Engineering Flashcards
What is genetic engineering
changing the genotype of an organism by transferring genes from one species to another
What is selective breeding
- used to domesticate animals and plants
- all you need is to be able to select and breed the next generation
- you can wait for useful mutations to arise by chance or you can cross breed 2 closely related species
What is gene cloning
moving a single gene from one organism to another
What are the 2 reasons to use genetic engineering to move genes around
- to make an organism express a new phenotype
2. to understand how the gene works
What do beta islet cells do in diabetes
- in pancreas
- sense high levels of glucose and release insulin which tells body to absorbs glucose
- glucagon is released when there is too little glucose
What causes diabetes
beta islet cells are destroyed by an autoimmune rxn so now the body can’t regulate glucose level
Steps to integrating a new gene into an organism
make gene, insert into plasmid and then transform into bacteria
Since bacteria can’t splice, genes without introns are needed how to you make these
make a DNA copy of the mRNA which has already been spliced
How do you make a DNA copy of already spliced mRNA
use reverse transcriptase enzyme from retrovirus
What is the DNA copy called
cDNA
What is a vector
plasmid you are using to carry the gene you are cloning
How do you get the vector to carry your gene
cut plasmid so its linear, and then join end of cDNA to end of plasmid to make a circle
How do we cut DNA
restriction enzymes
How do restriction enzymes work
identify a stretch of about 6 bps of a sequence and cut both ends at this point
What is a restriction site
sequence of DNA recognized by restriction enzymes
What do restriction enzymes leave
ragged/overhanging/sticky ends
What is the advantage of sticky ends
the overhang will try to bond to a complementary overhang which make ligation rxn more efficient since sticky ends stay together
How does cDNA stick to plasmid vector
DNA ligase (phosphodiester bonds)
What are the basic steps to gene cloning
- cut up plasmid vectors with restriction enzymes
- mix with cDNAs and some ligase
- mix that with bacteria so bacteria takes it up through transformation
- then we have bacteria each with a plasmid containing different cDNA
How do we know which bacteria have been transformed and which haven’t
put a gene on the bacteria that will show a specific phenotype on transformed bacteria and look for it (usually antibiotic resistance gene)
What are transformation markers
the special phenotype you look for
What is a cDNA library
allowing bacteria to grow and divide creating a little colony because bacteria represents a library of all of the mRNA (in cDNA form that can be made in a cell)
What is a DNA probe
if you know the amino acid of the protein encoded by the gene you are cloning you can make one
- it’s a piece of DNA that helps you find complementary DNA
- look for radioactivity to find out which colony the probe is stuck to
What is an expression vector
plasmid that will let the gene be expressed
How do we make the gene be expressed
give it a promoter and terminator sequence so that the bacterial RNA polymerase will recognize it as a gene
-put it in an expression vector (same process as putting into regular vector)
How do we know if our plasmid has the right number of inserts
gel electrophoresis
How does gel electrophoresis work
DNA is negatively charged and will move through an electric field toward the positively charged electrode (cathode). If you force the DNA to move through a thick tangle of agarose fibers (long strings of sugars) the bigger pieces will have a greater tendency to get stuck and will move more slowly. So if we
purify DNA from several transformed colonies of bacteria, run each on a gel, stain the DNA with a fluorescent dye, we can find the DNA that is the right size
What is the location of the restriction site into which you cloned your gene
is between the promoter, which consists of the lac promoter and the lac operator (but not the genes they control), and a terminator
True or False: lactose can regulate the gene
true
True or False: cystic fibrosis is a mendellian trait
true
is cystic fibrosis the most common autosomal disease among caucasians
YES, its recessive though
What is cystic fibrosis characterized by
respiratory problems, digestive issues, sterility inability to get rid of mucus, saltiness, mucus too dry
How can we figure out what causes cystic fibrosis
- find mutated gene and figure out what it encodes
2.sequence of protein can help us figure out what gene does
(gene mapping) gets you to a group of genes not a single one
What are restriction fragment length polymorphisms (RFLPs)
- variations in the sequence of a restriction site (like alleles kinda)
- presence of them in DNA results in presence or absence of a restriction site
- they segregate in a simple mendellian fashion
- we can assay the presence or absence of several restriction sites
- can be mapped like any allele
- result in new band lengths in PCR
How do you find RFLPs
LPs by looking for changes in the size of resulting DNA fragments after digesting genomic DNA with restriction enzymes
How do you test for the presence of a specific RFLP
polymerase chain reaction
What is the polymerase chain reaction
a method of producing large quantities of a short stretch of DNA from a known template DNA molecule
- uses DNA polymerase
- uses taq polymerase that loves hot temps
What is bioinformatics
finding genes using a computer
What are expressed sequence tags ESTs?
another way to find genes
-look for cDNAs and find genes that encode them
So what is cystic fibrosis caused by
- a mutation in the gene encoding a chloride channel that regulates the flow of chloride across the plasma membrane of epithelial cells. Specifically the flow of chloride into the lung
- When chloride isn’t regulated correctly, mucus becomes sticky and bacteria are able to grow
Is the cystic fibrosis allele beneficial
ya, heterozygous individuals for the allele are protected from typhoid fever (bacteria are not internalized efficiently)
