Chapter 19 Flashcards
Recombinant DNA Technology
Set of molecular techniques for locating, isolating, altering, combining, and studying DNA segments; also commonly called genetic engineering
Restriction Enzymes (or Restriction Endonuclease)
Enzymes that recognize particular base sequences in DNA and make double stranded cuts (restriction sites) nearby
Gel electrophoresis
Technique for separating charged molecules (such as proteins or nucleic acids) into fragment sizes
Ethidium Bromide
It has a role as a DNA intercalculator, inserting itself inbetween the base pairs in the double helix (don’t have to remember this part rn)
- basically works with gel electrophoresis under UV light to reveal fluorescent bands that shows the various DNA bands separated by size
PCR
PCR (polymerase chain reaction)
a laboratory nucleic acid amplification technique used to denature short segments of DNA or RNA sequences using DNA polymerase
Taq DNA Polymerase
DNA polymerase commonly used in PCR reactions due to its thermostability. Isolated from the bacterium thermus aquaticus, the enzyme is stable at high temperatures so it is not denatured during the strand-separation step of the cycle
Melting temperature
The temperature at which half of the DNA duplexes denatures
DNA fingerprinting
used in forensic testing or paternity testing, uses PCR to amplify micro satellite
Real-Time PCR
real-time PCR (qPCR) modification of PCR used to measure starting nucleic acids; DNA is simplified and measured as reaction proceeds
Gene-Cloning
insertion of DNA fragments into bacteria in such a way that the fragments will be stable and copied by bacteria (ex. E. coli)
Cloning vector
Stable, replicating DNA molecule to which a foreign DNA fragment can be attached for transfer to a host cell
DNA ligase
Enzyme that catalyzes the formation of a phosphodiester bond between adjacent 3’ - OH and 5’ phosphate groups in a DNA molecule
competent E. Coli
The ability of E. coli to take up DNA from the environment (to be transformed)
transformation
mechanism by which DNA found in the environment is taken up by a cell. after transformation recombination may take place between the introduced genes and the cellular chromosomes
CRISPR Cas genome editing
A method of splicing DNA to insert a donor, DNA CAS protein is expressed from streptococcus which uses S9RNA that has 20 nucleotides that are complimentary to target DNA. PAM is recognized by every CRISPR CAS 9 and occurs every 8bp
microsatellite DNA (STRs)
Short tandem repeat sequences found at many loci in a human genome
Sanger (dideoxy) sequencing
a technique for DNA sequencing based upon the selective incorporation of chain-terminating dideoxynucleotides (ddNTPs) by DNA polymerase during in vitro DNA replication.
4 Key Innovations of Molecular Genetics since discovery of three-dimensional DNA structure by Watson, Crick, Franklin and Wilkins:
- Development of recombinant DNA technology
- The invention of the polymerase chain reaction
- Development of quick methods to determining DNA sequences
- Engineering of CRISPR Cas System
Biotechnology
Use of biological processes, particularly molecular genetics and recombinant DNA technology to produce products of commercial value
Molecular Genetics
techniques used to study biology at the level of DNA, proteins, and other molecules; recombinant DNA technology is an important component of molecular genetics
Most recognition sequences (or restriction sites) are how many bp long?
4-8 bp and are “palindromic” that read the same 5’ to 3’ on the two complementary DNA strands
Overhanging sticky or cohesive ends
complementary fragments that can spontaneously pair to rejoin DNA fragments that have been cut with the same restriction enzymes.
can have either 5’ or 3’ strand overhanging ends.
HindIII Example Cohesive Cut
5’-A|AGCTT- 3’
3’-TTCGA|A- 5’
HindIII cuts the sugar-phosphate backbone of each strand at the point indicated by the line, generating in this example fragments with short, single-stranded overhanging 5’ ends or cohesive ends:
5’-A AGCTT- 3’
3’-TTCGA A- 5’
Blunt Fragments Example
PvuII cuts in the middle of its recognition sequence, and the cuts on the two strands are directly opposite each other, producing blunt-ended fragments.
5’-CAG|CTG-3’
3’-GTC|GAC-5’
5’-CAG CTG-3’
3’-GTC GAC-5’
In gel electrophoresis DNA migrates towards the positive pole because….
Because DNA has a strong negative charge due to it’s phosphate groups
How does gel electrophoresis separate DNA by size?
Gel hinders DNA mobility, smaller DNA fragments will migrate faster because their mobility will be less hindered than larger fragments
What is needed for two cohesive fragments to be ligated together?
Both need the same 3’ or 5’ overhanging ends
The single stranded parts need to be complementary
size of overhang has to be the same
Can two blunt ends DNA fragments be ligated (idk if ligated is a word but whatever) together? why?
Yes, it would need two enzymes that leave blunt ends and there is no need for complementary ends (matching sequences)
What are the two ways to amplify DNA?
PCR (polymerase chain reaction)
and
Gene cloning (inserting gene/DNA segment into plasmid, introducing E. coli, E. coli then replicates)
PCR Cycle Steps
- Denaturing 94C: requires high temp, close to boiling/strands separate
- Annealing 55C: primer (single strand short DNA) anneals to base pair with complementary sequence
- Extension 72C: polymerase can now extend/add nucleotides to 3’ hydroxyl and synthesize DNA
Each cycle number of DNA molecules double
(note: don’t need to memorize temps but given for example but like idk do whatever u want just know that denaturing is always the highest)
1kb is equal to how many bp?
1000bp (and 500bp would be like 0.5kb)
Melting temperature is higher of a primer during PCR when?
tm influenced by: length of primer and *GC (base pair) content
*GC pairs have more hydrogen bonding - when there is more hydrogen bonding between the primer and template DNA tm is higher
Gene cloning steps
- Digest - gene of interest is cut using restriction enzymes and plasmid is digested. plasmid is then cut using same restriction enzyme for complementary sticky ends
- Ligate - cut and glue together vector with DNA ligase
- Transform - ligation mixture goes into competent E. coli
- Select - E. coli plasmid contains DNA of interest and will grow and replicate
3 important features of a cloning vector
an origin of replication where DNA replication starts
DNA restriction sites
selectable marker (so e.coli cells can be distinguished between with and without plasmid
How does the Dideoxy Sequencing (Sanger method) result in DNA strands with different lengths?
Replication is terminated when a specific base is encountered resulting in DNA strands of different lengths
3 considerations for performing PCR
melting temperature - consider melting temp when choosing primer
high error rate - taq does not have proofreading 3’ to 5’ exonuclease
extremely sensitive - contamination could be an issue
Concept Q: DNA fragments that are 500 bp, 1000 bp, and 2000 bp in length are separated by gel electrophoresis. Which fragment will migrate farthest in the gel? Why?
500bp because smaller fragments migrate faster through gel
What is needed to perform PCR?
DNA Template
DNA Primer
dNTPs
Buffer
Thermal cycler
