Chapter 21 - genetic technologies and genomics Flashcards
1
Q
Recombinant DNA technology
A
- use of laboratory techniques to bring together fragments of DNA from multiple sources
2
Q
gene cloning
A
- the process of making multiple copies of a particular gene
- two purposes:
1. make large amounts of a specific gene’s DNA- study DNA directly
- use the DNA as a tool
- to make a large amount of the gene product
- study the structure and function of a protein
- other uses such as medicine
3
Q
genomics
A
- the molecular analysis of the entire genome of a species
4
Q
3 steps of gene cloning
A
- source vsctor DNA and chromosomal DNA as starting materials
- insert gene of interest into vector
- introduce a recombinant vector into a host cell that doesn’t already have a vector. The host cell takes up one vector and then copies the vector and divides to produce many cells
5
Q
vector DNA
A
- carrier for the DNA segment that gets cloned
- comes from plasmids or viruses
- can replicate once introduced into a living cell
6
Q
plasmids
A
small circular pieces of DNA found naturally in many strains of bacteria
7
Q
viral vectors
A
derived from viruses which infect living cells and propagate themselves using the host cell’s machinery
8
Q
recombinant vector
A
- contains plasma and the gene of interest
- made by opening up vector and inserting the gene of interest
- vector may recircularize without GEI insertion
9
Q
what is the importance of the location at which the GEI is cut
A
- the sequence on the overhang makes sure it attaches to the right place on the genomic DNA
10
Q
restriction enzymes
A
- cut DNA during gene cloning (step 2)
- made naturally by bacteria to protect against bacteriophages
- cut at specific restriction sites
11
Q
restriction sites
A
- sequence of DNA that binds a specific restriction enzyme
- most are palindromic
- overhangs are sticky because the complementarity causes them to attract each other
- cut in different places on the two sides of the DNA
12
Q
what is one con of the second step of gene cloning?
A
- vectors may recircularize without insertion of GEI
13
Q
DNA ligase in gene cloning
A
- covalently bonds the GEI into the vector DNA to create the recombinant vector
14
Q
selectable marker
A
- makes sure that the plasmid was taken up by the cells
- a gene whose presence can allow organisms such as bacteria to grow under a certain set of conditions
- ex: an antibiotic-resistance gene is a selectable market that can allows bacteria to grow in the presence of the antibiotic
- amp^R is the most common selectable marker
15
Q
amp^R
A
- most common selectable marker
- ampicillin resistance gene
- codes for beta-lactamase that degrades ampicillin, which normally kills bacteria
- growth of bacteria on ampicillin plates indicates that amp^R is present
- only cells that picked up the inserted plasma can grow
16
Q
LacZ system
A
- the LacZ gene is built into the vector to eliminate recircularized empty vectors
- codes for beta-galactosidase which cleaves colorless X-Gal into a blue dye
insertion of cloned DNA disrupts lacZ gene - bacteria with recircularized plasmids form blue colonies
- bacteria with recombinant vectors will form white colonies
17
Q
DNA library
A
- collection of many recombinant vectors each with a fragment of chromosomal DNA
- treatment of chromosomal DNA with restriction enzymes yields tens of thousands of different fragments
18
Q
genomic library
A
- type of DNA library with inserts derived from chromosomal DNA
19
Q
cDNA library
A
- uses reverse transcriptase to make complimentary DNA (cDNA) from mRNA
- lacks introns, simpler to use
- only genes that are expressed, more concise
20
Q
electrophoresis
A
- technique used to separate macromolecules on a gel
- can separate DNA or proteins
- often used to evaluate the results of a cloning experiment
- separate based on charge, size/length, and mass
- gel is set in aqueous solution so that charges can move through it and move the DNA
- denser and more massive molecules are more restricted and don’t move as far
21
Q
4 reagents of PCR
A
- DNA
- primers (forward and reverse)
- dNTPs (deoxynucleoside triphosphates)
- taq polymerase
22
Q
taq polymerase
A
- heat-stable form of DNA polymerase
- need to use taq because DNA is denatured using heat and human polymerase won’t work in high temps
- comes from bacterium called thermos aquatics that lives in hot springs
23
Q
polymerase chain reaction
A
- goal is to make many copies of DNA in a defined region
- doesn’t require vectors or host cells
- DNA runs through repeated cycles of denaturation, annealing and synthesis
24
Q
three steps of PCR
A
- denaturation - template DNA is heated and denatured into single-stranded molecules
- primer annealing - temperature is lowered and the primers bind to specific sites in the template DNA
- primer extension - temp is slightly raised and taq polymerase uses dNTPs to catalyze the synthesis of complementary DNA strands
25
Q
DNA sequencing
A
refers to a procedure that is aimed at determining the base sequence of DNA
26
Q
dideoxynucleoside triphosphate (ddNTPs)
A
- like dNTPs but are missing the 3’ -OH, so it can’t attach to the next base and causes the chain to terminate
27
Q
mapping of genome
A
- step in genomics involving the determination of the complete DNA sequence
- provides a detailed description of the organisms genome at a molecular level
28
Q
functional genomics
A
the study of the expression of a genome
29
Q
Dideoxy chain-termination method (dideoxy sequencing)
A
- DNA polymerase makes complementary strand to the single stranded template DNA until it reaches a ddNTP
- DNA chains of various lengths are created
- electrophoresis separates the chains by length
- the DNA sequence can be read by determining which base is at the end
- done in a test tube so there aren’t other types of DNA polymerases that go back and fix mistakes
30
Q
components of dideoxy sequencing
A
- many copies of the single stranded template DNA
- primers that bind to the primer-annealing site
- lots of all four regular dNTPs
- low concentration of all four ddNTPs tagged with a different fluorescent molecule
- DNA polymerase
31
Q
DNA microarray
A
- small slide on which an array of spots contains many different known sequences of single-stranded DNA
- used to monitor the expression of thousands of genes simultaneously
- goal is to find out which genes are transcribed into mRNA in a particular sample of cells
32
Q
CRISPR-Cas technology
A
- an experimental technique to introduce mutations into genes
33
Q
sgRNA
A
- single guide RNA
- single RNA in which the tracrRNA and crRNA are linked together
- spacer region of sgRNA designed to be complementary the DNA of a target gene to be mutated
- sgRNA binds to Cas9, guides it to the target genes
- Cas9 makes a double stranded break
34
Q
two different DNA repair events in crispr-cas technology
A
- end joining may lead to a small deletion
- if the researcher adds donor DNA homologous to the break region a specific mutation can be put into the gene
