Biotechnology Part 2 Flashcards
what is DNA fingerprinting?
a method that identifies an individual based on the patterns formed from the variations in the genetic code
what is tandemly repetitive DNA?
Also known as variable number tandem repeats (VNTRs) they are idetical DNA sequences repeated in series (tandem)
short tandem repeats (STR)
a subcategory where repeat length is short - most commonly 4 bases long
Polymorphic
- many forms - many alleles - more than the standard 2 alleles
what is a DNA fingerprinting method?
- 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
why are noncoding regions used?
coding regions are too similar between individuals and even species
paternity inclusion
means he can’t be excluded from being a possible paternity but no CONFIRMATION
gene cloning
making multiple copies of a single gene by using in vivo amplification
what are the 3 steps of gene cloning?
- forming recombinant DNA
- transformation (followed by many cell devisions)
- selection
recombinant DNA
genes from two different sources (often different species) combined into one molecule
episome
genetic elements that can exist either as a plasmid or as part of the bacterial chromosome
describe step 1, forming recombinant DNA
- 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
plasmid
small, circular self-replicating pieces of DNA with a small number of genes that incorporate themselve into the bacterial chromosome
why are plasmids advantageous?
- not required for bacterial cells to survive in normal conditions
- when there are stresses, genes on plasmids can confer advantages
- increases genetic variation
what is a cloning vector?
artificially manipulated plasmid into which the gene of interest is introduced
what does the cloning vector contain?
- ori
- promoter
- restriction sites / cloning site
what does the ori do in a vector?
allows the plasmid to replicate in the host cell
cloning site
- where the gene of interest will be inserted (ligated)
- where transcription can occur because contains an upstream promoter
describe step 2 - transformation gene cloning
- 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
naked DNA
- DNA not inside a cell
- donor cell can be dead or nonexistant
what is a natural method of transformation?
- some bacteria have surface proteins that recognize and transport DNA from closely related species
what is an artificial method of transformation?
transformation may occur when bacteria is subjected to treatments that make the cell wall permeable
what are specific methods of artificial transformation? Describe them.
- Chemical: Cold CaCl2 treatment followed by heat shocking
- Eletroporation: cell are shocked with an electric current to create holes in the bacterial membrane
describe DNA amplification in vivo
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
bacterial product: no vector
cause?
no transformation or transform without vector
bacterial product: empty vector
cause?
transform with unsuccessful ligation
bacterial product: recombinant
cause?
transformation and successful ligation
what is selection in gene cloning?
indentifying colonies of bacteria containing the recombinant DNA
what is plating in gene cloning?
taking a sample of the bacteria and growing them on plates
plates have an agar medium containing…..
antibiotics
X-gal
antibiotic resistance gene (ampR)
allows cells to be resistance to ampinilin - an antibiotic
how are bacteria selected?
bacteria cells that properly transformed also have the antibiotic resistance so they will survive after being exposed to the virus
what does beta galatosidase (LacZ) do?
enzyme produced will change a clear substrate called X-gal into a blue product
ligation
joining different nucleic acids usually involving ligase
genome
an organisms’ complete set of DNA
human genome project
an international collaborative research effort to sequence and map all genes in human beings
what sequencing approach did Celear Genomics (private company headed by Craig Venter) use?
shotgun
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
What sequence approach did PUbliv research institutions worldwide use?
small scale shotgun
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
What id Fredrick Sanger develop?
the dideoxy termination sequencing method
What is a dideoxyribonucleotide (ddNTP)?
a ribonucleotide missing two oxygens
where are the two oxygens msising from? their location
both at the 2’ and 3’ position position
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
What materials are used in sanger sequencing?
- DNAP
- Primer (radioactive)
- template DNA
- dNTPs
- ddNTPs
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
what are the 5 main experiments that determine DNA as the genetic material?
- Miescher
- Griffith
- Hammerling
- Avery, McCarty, MacLeod
- Hershey, Chase
What did Fridrich Miescher discover?
DNA
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
describe nuclein
- present in enery cell type tested
- high is phosphorous
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
what did Griffith study?
bacterium that causes pneunomia
- streptococcus pneumoniae
Describe a Rough colony/bacteria
- benign
- lacks a protective capsule
recognized and destroyed by host’s immune system
describe the Smooth bacteria
- virulent
- has a polysaccharide capsule that prevents detection by hosts’s immune system
- kills the host
What did Griffith define as the transformation observed phenomenon?
- change in a cell’s function by the transfer of an unknown substance
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
What species did hammerling use?
single cellular green alga Acetabularia
what were the 3 distinct parts of the acetabularia?
- foot containing the nucleus
- stalk
- cap
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
What did Avery determine?
that DNA is a hereditary material
What question did Hershey and Chase ask?
- which viral component is resposible for reprogramming, DNA or protein?
What materials were involved in the Hershey chase experiments?
- bacteriophage
- radioactive sulfur to label proteins
- radioacitive phosphorus to label DNA
describe the centrifugation of the Hershey Chase
supernatent were free phage and phage parts and the pellet was the bacterial host cell
what happened in the Hershey chase experiment when radioactive sulfur was added?
the supernatent was radioactive
what happened when radiactive phosphorous was added?
the pellet was radioactive
what did Beadle and Tatum propose?
that genes were responsible for producing enzymes
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
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
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
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
describe the first step in the beadle and tatum experiment - making mutants
- treated mold with x rays to mutate the DNA
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
what is a complete media?
contains eberything needed for growth without mold bacing to synthesize it
what does a complete media contain?
- salt
- sugar
- all vitamins
- all amino acids
describe a minimal media?
something that only has what is essential for growth
what does a minimal media contain?
- salt
- sugar
- biotin
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
describe a supplemented media?
only has what is essential for growth plus one nutrient
what are the contents of a supplemented media?
- salt
- sugar
- vitamin
- biotin
- 1 nutrient
what was the result of the beadel and tatum rationale?
malfunctioning enzyme was due to mutations in genes
what is the one gene, one enzyme hypothesis?
the function of a gene is to dictate the production of a specific enzyme
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
