Biotechnology Part 2 Flashcards
1
Q
what is DNA fingerprinting?
A
a method that identifies an individual based on the patterns formed from the variations in the genetic code
2
Q
what is tandemly repetitive DNA?
A
Also known as variable number tandem repeats (VNTRs) they are idetical DNA sequences repeated in series (tandem)
3
Q
short tandem repeats (STR)
A
a subcategory where repeat length is short - most commonly 4 bases long
4
Q
Polymorphic
A
- many forms - many alleles - more than the standard 2 alleles
5
Q
what is a DNA fingerprinting method?
A
- PCR - takes fragment of interest at a specific loci on the chromosome
- PCR amplifies the fargment
- uses gel electrophoresis to separate PCR products by size and visualize the DNA fragmentsk
6
Q
why are noncoding regions used?
A
coding regions are too similar between individuals and even species
7
Q
paternity inclusion
A
means he can’t be excluded from being a possible paternity but no CONFIRMATION
8
Q
gene cloning
A
making multiple copies of a single gene by using in vivo amplification
9
Q
what are the 3 steps of gene cloning?
A
- forming recombinant DNA
- transformation (followed by many cell devisions)
- selection
10
Q
recombinant DNA
A
genes from two different sources (often different species) combined into one molecule
11
Q
episome
A
genetic elements that can exist either as a plasmid or as part of the bacterial chromosome
12
Q
describe step 1, forming recombinant DNA
A
- gee of interest is inserted into a bacterial plasmid
- restriction enzyme digest plasmid and gene of interest so they have similar ends
- hybridization of matching sticky ends on gene of interest and plasmid
- DNA ligase seals gene of interest with plasmid
13
Q
plasmid
A
small, circular self-replicating pieces of DNA with a small number of genes that incorporate themselve into the bacterial chromosome
14
Q
why are plasmids advantageous?
A
- not required for bacterial cells to survive in normal conditions
- when there are stresses, genes on plasmids can confer advantages
- increases genetic variation
15
Q
what is a cloning vector?
A
artificially manipulated plasmid into which the gene of interest is introduced
16
Q
what does the cloning vector contain?
A
- ori
- promoter
- restriction sites / cloning site
17
Q
what does the ori do in a vector?
A
allows the plasmid to replicate in the host cell
18
Q
cloning site
A
- where the gene of interest will be inserted (ligated)
- where transcription can occur because contains an upstream promoter
19
Q
describe step 2 - transformation gene cloning
A
- transforms recombinant DNA into bacterial cell
- all bacteria multiplies the gene of interest is also replicated
- alteration of bacterial DNA by uptake of foreign DNA
20
Q
naked DNA
A
- DNA not inside a cell
- donor cell can be dead or nonexistant
21
Q
what is a natural method of transformation?
A
- some bacteria have surface proteins that recognize and transport DNA from closely related species
22
Q
what is an artificial method of transformation?
A
transformation may occur when bacteria is subjected to treatments that make the cell wall permeable
23
Q
what are specific methods of artificial transformation? Describe them.
A
- Chemical: Cold CaCl2 treatment followed by heat shocking
- Eletroporation: cell are shocked with an electric current to create holes in the bacterial membrane
24
Q
describe DNA amplification in vivo
A
once the bacteria are transformed, they are transferred to and grown in a liquid medium so the total number of bacterial cells and DNA increases
25
bacterial product: no vector
cause?
no transformation or transform without vector
26
bacterial product: empty vector
cause?
transform with unsuccessful ligation
27
bacterial product: recombinant
cause?
transformation and successful ligation
28
what is selection in gene cloning?
indentifying colonies of bacteria containing the recombinant DNA
29
what is plating in gene cloning?
taking a sample of the bacteria and growing them on plates
30
plates have an agar medium containing.....
antibiotics
X-gal
31
antibiotic resistance gene (ampR)
allows cells to be resistance to ampinilin - an antibiotic
32
how are bacteria selected?
bacteria cells that properly transformed also have the antibiotic resistance so they will survive after being exposed to the virus
33
what does beta galatosidase (LacZ) do?
enzyme produced will change a clear substrate called X-gal into a blue product
34
ligation
joining different nucleic acids usually involving ligase
35
genome
an organisms' complete set of DNA
36
human genome project
an international collaborative research effort to sequence and map all genes in human beings
37
what sequencing approach did Celear Genomics (private company headed by Craig Venter) use?
shotgun
38
Describe the shotgun sequencing approach
radomly breaking up DNA sequences into lots of small pieces, sequencing fragmets and reassembling them by looking for regions of overlap
39
What sequence approach did PUbliv research institutions worldwide use?
small scale shotgun
40
describe the small scale shotgun sequencing approach
same as shotgun but markers are used a regular intervals in the genome to make it easier to reassemble the sequence
41
What id Fredrick Sanger develop?
the dideoxy termination sequencing method
42
What is a dideoxyribonucleotide (ddNTP)?
a ribonucleotide missing two oxygens
43
where are the two oxygens msising from? their location
both at the 2' and 3' position position
44
How do dideoxynucleotides work?
the 3'OH that is needed to react with the phosphate group on the 5' end of the next nucleotide to form a phosphodiester bond is no longer there so the chain can't continue
45
What materials are used in sanger sequencing?
- DNAP
- Primer (radioactive)
- template DNA
- dNTPs
- ddNTPs
46
describe the difference between modern sequencing and the original Sanger method?
- 4 differently coloured fluorescent dyes used to label the 4 different ddNTPs in a single test tube so it can be run on one lane on a gel electrophoresis
-
47
what are the 5 main experiments that determine DNA as the genetic material?
- Miescher
- Griffith
- Hammerling
- Avery, McCarty, MacLeod
- Hershey, Chase
48
What did Fridrich Miescher discover?
DNA
49
What did Friedrich Miescher do in his experiments?
- collected white blood cells from pus
- lysed cells and isolated nuclei
- where he found a substance he called nuclein
50
describe nuclein
- present in enery cell type tested
- high is phosphorous
51
What was the DNA vs Protein debate?
- is DNA or protein the most important hereditary molecule?
- money on protein since had obvious function and 20 amino acids to be combined in dif ways
- whereas DNA only 4 in same pattern bc of Levene
52
what did Griffith study?
bacterium that causes pneunomia
- streptococcus pneumoniae
53
Describe a Rough colony/bacteria
- benign
- lacks a protective capsule
recognized and destroyed by host's immune system
54
describe the Smooth bacteria
- virulent
- has a polysaccharide capsule that prevents detection by hosts's immune system
- kills the host
55
What did Griffith define as the transformation observed phenomenon?
- change in a cell's function by the transfer of an unknown substance
56
What is the current definition of the transformation observed phenomenon?
a change in genotype and phenotype due to assimilation of external DNA by a cell
57
What species did hammerling use?
single cellular green alga Acetabularia
58
what were the 3 distinct parts of the acetabularia?
- foot containing the nucleus
- stalk
- cap
59
What were the two hammerling experiments?
- removed the cap vs the foot from the plant and observed what regrew
- grafted the stalk of one alga type onto the base of another alga type
60
What did Avery determine?
that DNA is a hereditary material
61
What question did Hershey and Chase ask?
- which viral component is resposible for reprogramming, DNA or protein?
62
What materials were involved in the Hershey chase experiments?
- bacteriophage
- radioactive sulfur to label proteins
- radioacitive phosphorus to label DNA
63
describe the centrifugation of the Hershey Chase
supernatent were free phage and phage parts and the pellet was the bacterial host cell
64
what happened in the Hershey chase experiment when radioactive sulfur was added?
the supernatent was radioactive
65
what happened when radiactive phosphorous was added?
the pellet was radioactive
66
what did Beadle and Tatum propose?
that genes were responsible for producing enzymes
67
why was using bread mold a good choice for beadle and tatum?
aka neurospora crassa
- simple organism
- short life cycle
- only requires few biological substances so it could grow on minimal media
68
what did beadle and tatum hypothesize??
- must be able to synthesize all the other vitamins and other essential nutrients on their own
- have enzymes that convert the simple substances into essential nutrients needed for growth
69
what did Beadle and Tatum do in their experiment?
- mutated the genes in the mold
- looked for mutations that affected the mold's ability to make essential nutrients
70
what were the three steps in the experimental procedure for beadle and tatum?
- X rays to make mutants
- selection to isolate the mutants
- supplementation to identify the mutants
71
describe the first step in the beadle and tatum experiment - making mutants
- treated mold with x rays to mutate the DNA
72
describe the second step in the beadle and tatum experiment - isolating mutants
- looked for mutations where mold grew in complete enriched media and could no longer grow in in minimal media
73
what is a complete media?
contains eberything needed for growth without mold bacing to synthesize it
74
what does a complete media contain?
- salt
- sugar
- all vitamins
- all amino acids
75
describe a minimal media?
something that only has what is essential for growth
76
what does a minimal media contain?
- salt
- sugar
- biotin
77
describe the third step in the beadle and tatum experiment - identifying mutants
grew the mutant in minimal media pluts vitamins OR nucleic acid, if the mutant grow it laced the ability to make that nutrient
78
describe a supplemented media?
only has what is essential for growth plus one nutrient
79
what are the contents of a supplemented media?
- salt
- sugar
- vitamin
- biotin
- 1 nutrient
80
what was the result of the beadel and tatum rationale?
malfunctioning enzyme was due to mutations in genes
81
what is the one gene, one enzyme hypothesis?
the function of a gene is to dictate the production of a specific enzyme
82
why would one gene, one enzyme be inaccurate?
- not all enzymes are proteins
- one protein may have more than one subunit each one a separate polypeptide
- some genes do not produce polypeptide
- one gene may produce multiple polypeptides due to splicing
