Chapter 19 - Recombinant DNA Technology Flashcards
The first recombinant DNA experiments were done in the __s. It combined DNA from __ sources to create a __ DNA molecule.
70s.
different; novel/new
__ means DNA from different sources is combined to create a novel DNA molecule. Also known as genetic __.
Recombinant.
Engineering
Prior to recombinant tech, the genotype had to be __ from the __. Mutations could only be created __; and because of their complexity, __ organisms could not be studied with the old techniques.
inferred.
randomly; eukaryotic
One of the challenges of studying genetics at the molecular level (as opposed to __ the genotype) is finding the gene of interest is like finding a __ __ __ __. Once found, producing enough of the gene and gene __ would be an enormous undertaking.
inferring; needle in a haystack.
product
A fundamental requirement for DNA manipulation at the molecular level is __ and __ DNA fragments. This is made possible by __ enzymes called __ __ (aka __ __).
cutting and joining.
bacterial; restriction endonucleases; restriction enzymes.
The biological purpose of restriction enzymes is to make __-__ cuts in DNA of viruses and causing the foreign DNA to __.
The bacterial DNA isn’t targeted because it is “__” by a specific pattern of ___.
double-stranded; degrade.
“marked”; methylation
Brake down the nomenclature of the following __ enzyme:
EcoR1
Restriction;
Eco = E. coli (source);
R1 = 1st restriction endonuclease/enzyme discovered.
Most __ enzymes are able to recognize small sequences about 4 - 6bp in length. The recognition sequence is always a __ (read the same way __ and __). They can be bisected __ and __.
restriction.
palindrome; forward; backward.
horizontally; vertically
Complete the following recognition sequence for restriction enzyme XyzR2: 3’ TTC
a) 5’ GAA
b) 5’ AAG
c) GAA 5’
d) more than one of the above
d) more than one of the above
b) 5’ AAG AND c) GAA 5’
What are the 2 types of cuts made by restriction enzymes? What are the ends of one of them called, and why does it have that name?
(1) Blunt end cuts (no single-stranded ends)
(2) Staggered cuts (single-stranded ends).
Staggered cuts make “sticky” ends or “cohesive” ends. They’re so named because the ends are complimentary and will ‘stick’ or reanneal if given the opportunity.
Blunt end vs. sticky end cuts: which is more advantageous?
blunt
DNA from different sources can be __ by the same __ enzyme to produce complimentary __.
digested; restriction; termini
PCR is DNA __ converted into a test-tube reaction. A fragment can be __ over 1B fold in one hour. It is the most __ method for DNA analysis. It can analyze DNA from a single __.
cloning.
amplified.
sensitive.
cell.
PCR is a chain reaction because it __ at every step.
doubles.
True PCR product is produced at the __ round of replication. But in general, after 30 cycles, DNA is amplified ^, or more than one billion fold.
3rd.
2^30
To order PCR primer, one needs to know the sequence of the __ of the target element.
Once ordered, DNA is __, primer is added, primer attaches to the __ of each strand, and new DNA is synthesized. Rinse and repeat.
termini.
denatured; termini
One of the problems to overcome in automating PCR was finding a __-__ DNA polymerase. The first used was __ from a thermophilic prokaryote, but it is an __-__ polymerase because it lacks __ activity. Subsequent polymerases with better __ have been since been discovered.
heat-stable.
Taq; error-prone; proofreading.
accuracy
What can real-time PCR do that automated PCR can’t do? Why is this valuable (i.e. what can it be used to test)?
Real-time PCR can quantitate the amount of PCR product.
It’s valuable because it can “measure” changes in gene expression - if you add mRNA from regular cells vs. cancer cells, the amount of PCR product is directly proportional to the amount of mRNA, so you can infer that the cancer cells are expressing more mRNA than normal cells.
Gene discovery is becoming more the result of “__ __” of extensive and rapidly growing DNA databases (the latin phrase “In __” has been coined). The field is called ___, but there are many other names like genomics, proteomes, transcriptomes, etc.
“data mining”; “In Silico”.
bioinformatics.
__ __ __ is a technique that uses a complimentary “probe” to determine the cellular or chromosomal location of the __ or __ product. Basically, it is in vivo ___ and __ of a biomolecule.
In situ hybridization; gene; gene product.
tracking and visualization
DNA __ exploits the highly-variable sequences among different individuals. These __ sequences do NOT have to be a gene; many are __ or __ __ __. The sequences of interest are amplified using __.
fingerprinting.
polymorphic; microsattelites; short tandem repeats (STRs).
PCR
The 1st method used for DNA fingerprinting was __ __ __ __ (RFLP). It uses __ enzymes that target small sequences (called __ sequences) along the length of the molecule. The __ enzyme “digests” each __ sequence creating DNA __ of varying ___. People vary in the number of __ sequences, so they will yield fragments that vary in __ and __. These fragments will separate according to their __ during electrophoresis.
restriction fragment length polymorphism.
restriction; recognition.
restriction; recognition; fragments; lengths.
recognition, length; number.
size.
A RFLP is used to test 3 individuals. One person has banding found in pattern D; the second had banding found in pattern Q; and the third person has banding found in both patterns D and Q. what can you conclude about these individuals?
Person 1 is homozygous for pattern D; person 2 is homozygous for pattern Q; and person 3 is heterozygous for patterns D and Q.
Using __ or __ __ __ for DNA fingerprinting is more advantageous than using RFLPs because there are more “__” to work with.
microsattelites; short tandem repeats; ‘alleles’
