DNA Technology Flashcards
vectors
Things used to transport genes into cells
first vectors used were
plasmid vectors
plasmid vectors
contain only a few genes (non essential)
bacteria will take in plasmids from the environment
what takes in plasmids
only bacteria take in plasmids, not eukaryotes
virus vectors
viruses inject DNA into nucleus of host cell
We replace virus DNA with genes we want placed in a cell
used in gene therapy
example of what virus vectors treated
B in B disorder (no immune system, live in sterile environment)
virus put new gene in stem cells
stem cells returned to bone marrow
10 of 11 cured (3 later got leukemia)
restriction enzymes
Enzymes that cut DNA ONLY at specific sites
only cuts double stranded DNA
how do restriction enzymes cut at specific sites
Sites are identified by nucleotide patterns on both strand of DNA
Restriction enzymes were isolated from
Bacteria, which use them as primitive immune system to cut virus DNA
cDNA
complimentary DNA
why cDNA
used to put human gene in bacteria
bacteria can’t remove introns from mRNA transcripts so we remove introns from the gene before we give it to them
we let the human cell do transcript processing
then use mRNA as a template
making cDNA
- Get human mRNA for needed protein
- Add reverse transcriptase that bonds DNA nucleotides to RNA
this DNA is the cDNA
3.Add DNA polymerase to remove RNA nucleotides and replaces them with DNA nucleotides - End result = double stranded DNA
can be cut with restriction enzymes
& added to plasmids that bact. take in.
probe
used to located gene of interest, Short strand of DNA complementary to gene of interest, Tagged with radiolabel or tracer, binds to gene of interest which makes hybrid DNA
Hybrid DNA
Hybrid DNA = any DNA where the 2 sides of the double helix come from different sources
DNA isolation
isolate the gene you want from the rest of the genome
DNA isolation process
A. Use probes to label DNA B. Cut DNA using restriction enzymes C. Separate DNA using electrophoresis D. Use only DNA that includes the radiolabeled probe E.Reduces volume of unwanted DNA
electrophoresis
reduces volume of unwanted DNA
separates DNA by size
uses agar and electricity, DNA moves toward positive end, little pieces go the fastest
primer
A. Short, synthetic, single strand of RNA or DNA
B. Complimentary to DNA in front of gene of interest
C. Initiates DNA synthesis
PCR
polymerase chain reaction, makes thousands of copies
Step A for PCR
Mix DNA containing GoI with
1) primers for target gene
2) spare nucleotides (A, T, G, C)
3) DNA Polymerase from Taq polymerase which is a heat resistant enzyme
Step B for PCR
Step B : heat DNA strands to break H-bonds between base pairs and separate the strands
Step C for PCR
cool DNA so base pairs re-bond ( some bond to primer)
Step D for PCR
repeat whole process, 1) mix DNA with primers, nucleotides, and taq
2) heat to break H bonds
3) cool to bond to primer
4) repeat
Gel Electrophoresis-
separates DNA based on size of fragment
electrophoresis process
Samples of DNA cut with same restriction enzymes
Dye added to visualize DNA
Samples placed in wells of gel
Gel placed in liquid buffer (to run current through)
Electric current run through buffer & gel
DNA moves through the gel to positive end
smaller pieces move faster
Factors affecting DNA movement in gel electrophoresis
- More porous gel (less dense) speeds up movement
2. Higher current speeds up movement
gel analysis
A. DNA standard marker of known length are in lane 1 B. Measure distance each segment moved C. Graph distance vs size D.Best fit line E. Estimate unknown size
DNA fingerprinting
Different people have different lengths of DNA between genes
the Differences in human genes are in the
non-coding DNA between genes tandem repeats, the number of repeats varies, so the lengths of DNA varies
DNA fingerprinting uses
Crime scene investigations
Paternity tests
paternity tests
child gets all DNA from mom or dad, so Any fragment found in child must be in either Mom’s DNA or Dad’s DNA
DNA sequencing
Alter nucleotides so they will stop replication
DNA sequencing process
Label Nucleotides with fluorescent dye each base different color
Alter labeled nucleotides so they stop DNA replication
Add both labeled nucleotides and normal nucleotides to sample DNA
(Add modified A nucleotides to one sample,T to second, G to third, C to fourth)
Run PCR then electrophoresis
Order of segments indicates order of bases in sequence
Sanger Method (dideoxy method)
method for DNA sequencing, Uses Dideoxynucleotide to stop replication, named after Frederick sanger, won nobel prize in chemistry
reading DNA sequencing
Segments separated by size
Base that fluoresces is always the last base in line
GMO
genetically modified organism
genetic engineering
foreign genes added to organisms
GMO example (bt gene from Bacillus thuringiensis)
bt gene from Bacillus thuringiensis
a) bacterial protein toxic to insects b) used by WHO to kill mosquitoes c) used by organic farmers d) added to corn potato cotton so plant makes its own bt protein insecticide e) specific to borers & bollworm
bt mechanism
plant uses bt gene to make protein, insect eats protein, protein binds to receptors in gut, gut wall breaks down, only insects with receptor affected, studies show no harm to Monarchs
bt advantages
less pesticide, more specific delivery
transgenic
organism with foreign DNA
1) DNA microinjection into egg
2) all cells of organism have new DNA
