Ch. 10: Genetic engineering Flashcards
restriction enzymes
Endonuclease; cut/break phosphodiester bonds (in the backbone of DNA) at specific sequences; destroy DNA bc they think its a virus
-could be 4-6 nucleotides long; if not the exact sequence, they won’t cut it
-palindromes allow enzyme to cut through
-create restriction fragments (DNA pieces)
restriction fragments
DNA fragment resulting from the cutting of a DNA strand by a restriction enzyme (restriction endonucleases)
cDNA (Complementary DNA)
DNA created by using reverse transcriptase to synthesize DNA from RNA templates
nucleic acid hybridization
-when you have 2 strands of nucleic acids coming together & binding/forming hydrogen bonds (DNA, RNA etc.. as long as they are complementary sequences)
-can be used with oligoprobes (short DNA/RNA that is labelled with dye/something for detection)
gene probe
single-stranded short DNA fragment that has the exact sequence that’s complimentary to what you are looking for;
-got something so you can see it- radioactive or fluorescent
[a single-stranded sequence of DNA or RNA used to search for its complementary sequence in a sample genome.]
DNA sequencing
process of determining the exact order of nucleotides in a DNA segment ranging from small pieces > whole chromosomes/genomes.
The newest technologies are rapid machine-based methods with lasers/cams/microscopes that read fluorescent nucleotides on DNA strand
a common method is SANGER DNA sequencing
–need primers, TAQ DNA polymerase
involves electrophoresis and is based on the random incorporation of chain-terminating dideoxynucleotides by DNA polymerase
Dideoxunucleotide
chain-elongating inhibitors of DNA polymerase, used in the Sanger method for DNA sequencing. They are also known as 2’,3’
used to terminate growing DNA chains and create the subsets of truncated fragments in a sequencing reaction
recombinant DNA
-synthetic process- taking DNA from multiple sources and mixing them up; restriction enzymes used to make DNA.; makes immune treatments, hormones, enzymes, vaccines, enzymes used to make cheese…
- Isolate DNA you want [thru southern blot, PCR, reverse transcriptase, or restriction enzymes]
- Insert into vector plasmid [bacterias sees plasmids as being advantageous/helpful, so DNA inserted thru plasmid]
- insert into host
genetically engineered
also called genetic modification) is a process that uses laboratory-based technologies to alter the DNA makeup of an organism.
clones
colony of cells derived from a single cell by asexual reproduction.[units share identical characteristics]
plasmid vector
a DNA molecule that is used as a vehicle to carry a particular DNA segment into a host cell as part of a cloning or recombinant DNA technique.
transgenic
cells/organims into which foreign DNA has been introduced; includes organisms created through genetic engineering
gene therapy
introduction of normal functional genes into people with genetic disorders like sickle cell or cystic fibrosis;;; usually accomplished by a virus vector
DNA fingerprinting
method of identifying patterns of specific genetic markers unique to an individual for purpose of identification; used in forensics, medicine, microbial identification…
-old method- just using your DNA & gel & restriction enzymes as they are specific to every single person;;; checking RFLPS restriction fragment length polymorphism; requires a lot of DNA like blood, semen, cheek swab
-new method— using PCR; needs small amount of DNA; FBI uses this
what enzyme is used to join 2 pieces of DNA together into a single strand?
Ligase
A technique that separates DNA fragments of different sizes is
gel electrophoresis.
When patient tissues are combined with viruses carrying a needed, normal human gene, the technique is called
gene therapy.
Agrobacterium tumefaciens is used:
to genetically engineer plants
True or False: There is scientific evidence that GMOs are associated with some negative effects on human health.
false
Restriction enzymes are obtained from various species of:
bacteria.
CRISPR is used:
to genetically alter organisms
-easy, efficient way of genetic manipulation of any gene in any organism
Both DNA sequencing and polymerase chain reactions require special ________ to initiate synthesis of a new DNA molecule.
primers
The most common vector used in genetic engineering is:
plasmid
_________________ is often used in forensic science to distinguish one sequence of DNA from another by comparing the sequence of the strands at specific loci?
DNA fingerprinting
DNA strands can be cut at selected sequences by using enzymes called
restriction enzymes.
Transformation and transduction are methods used to introduce DNA into _______________
host cells.
What animals/produce has been genetically modified for human consumption?
- sugar beet, beets, soybeans, corn, cotton, papaya, squash,
- apples (prevents browning)
- salmon (grows 2x fast)
Geneticists can make complimentary DNA copies of RNA by using:
reverse transcriptases.
When DNA is heated to just below boiling (90°C–95°C), what happens?
the two DNA strands separate completely.
Which PCR step causes the denaturation of double-stranded DNA?
Heat target DNA to 94°C.
Amplification of DNA is accomplished by-
polymerase chain reaction.
The primers in PCR are-
short strands of DNA (oligonucleotides).
Nanopore sequencing enables sequencing of a DNA strand by measuring:
voltage changes through a membrane
The Southern Blot technique detects:
DNA.
during transformation, a cell takes up the:
plasmid
DNA polymerases used in PCR must remain active at:
very high temperatures.
When determining the sequence of nucleotides in an unknown sample of DNA, which method is used to sequence the DNA?
The Sanger Method
Reverse transcriptase
enzyme that reverses transcription; makes a DNA copy from RNA (reverse of transcription) = cDNA complementary DNA.
-uses mature mRNA (introns already spliced out, only exons)
-2 natural sources of this—viruses like HIV or in our cells when want to make long telomeres; (longer telomeres = longer life. They shorten as you age). Cancer cells also have reserve transcriptase turned on so they don’t die.;
genetic engineers use reverse transcriptase; took human gene for insulin which has introns>took mRNA and made cDNA copy>into e coli> made human insulin; still made in e coli today, lacking introns.
Explain the purpose and the process of a Southern blot.
-looking for a specific DNA sequence in a sample
-take a cheek swab/stool etc and extract all DNA, can cut with restriction enzymes and run through electrophoresis gel; transfer DNA to a membrane paper; convince DNA to move from gel to paper>ziplog bag> squirt in the probe [short DNA sequence needed] ;;; if probe sticks, the complementary gene sequence is there so u know the person has herpes/HIV/cancer gene etc…
Explain the purpose and the process of PCR.
-The purpose is to copy short sections of a specific DNA sequence; use test tube DNA, molecules, enzymes…
—discovered by Kary Mullis (while high on acid)
-Use for hospital diagnostics, paternity tests, genetic engineering, forensics, evolutionary studies, identifying random pieces of bone, research…
-Very sensitive, so can pick up and make billions of copies of few nucleic acids; highly prone to contamination from air even…
-At its core, involves 3 steps
Polymerase chain reaction 30x for PCR reaction- done thru temperature cycling
—Denaturation- heating to 94/95 C = separate DNA strand
—Annealing- primers attach to single strand molecule and find a complimentary sequence if it is present
—Extension- warmer- 72 c - DNA polymerase works and insert nucleotides 1 by 1 extending strand
How does one genetically modify a plant?
2 ways-
1) Agrobacterium- insert its DNA into a plant
2) use a gene gun - shoot pieces of DNA into plant cells
-used to make BT crops or Roundup ready crops
=Genetically modified foods -sugar beet, beets, soybean (oil), corn, cotton, papaya, squash, apples (prevents browning), salmon (grows 2x fast)
what are 2 ways a DNA helix can become separated?
- enzyme helicase
- heating it up - below water BP around 90-95c
=DNA denaturation
can a split, heated DNA helix reform?
yes, when it is cooled can reform if sequencing complement eachother= renaturation
DNA is negatively charged because of the-
negatively charged phosphate groups on the backbone
Gel electrophoresis
-a process whereby you separate DNA fragments based on their size
-gel is made of agarose (purified agar); use casting tray, comb, gel, make pockets called wells, salt water buffer allows electric current go thru gel; DNA will want to run to positive end; movement; shorter fragments go further & long ones get stuck
Oligonucleotides
short DNA or RNA molecules
Nanopore DNA sequencing
new DNA sequencing- very fast, uses reusable membrane, dna is ran over this membrane, enzyme un winds DNA > single strand > electical current used.. Not as accurate as Sanger but very rapid
transgenic animals
genetically modified animals- uses electric shock which disrupts cell membrane allowing recombinant DNA inside…or uses virus
-Salmon (FDA approved) grow 2x as fast, 1 year
-mosquitos (malaria resistant) designed to reduce malaria spread
