Chapter 19 - Molecular Genetics and Biotechnology Flashcards
What are the 4 key innovations in molecular genetics?
Recombinant DNA technology
Polymerase chain reaction
DNA sequencing
Genome editing systems
What are the 6 challenges with working at the molecular level?
Genes are minute
Small physical quantities
Stability of molecules
Complexity of genome (introns, etc.)
Single cell has millions of genes
Nucleotides can’t be seen
What is recombinant DNA technology?
AKA: genetic engineering
Take a segment of DNA and transfer it to another cell to study it
Allows us to locate, isolate, alter, combine, and study DNA fragments/genes
What does recombinant mean?
Taking DNA from two sources and putting them together
What are restriction enzymes and what is their role in recombinant DNA technology?
Role: Isolate DNA sequence
- Recognize specific nucleotide sequences and make double-stranded cuts at the specific restriction sites
- Produced by most eukaryotes
- 4-8 base pair cutters
- Sticky ends: have overhangs of nucleotides
- Blunt ends: no overhang
- DNA molecules cut with the same restriction enzyme have complementary sticky ends that pair if fragments are mixed together
- Shorter restriction enzymes may have a sequence that is seen more throughout the genome, which makes it cut more
What are the 3 parts of recombinant DNA technology?
Restriction enzymes - isolate DNA sequence
Visualizing DNA
Cloning vectors - DNA inserted into new source
What are the 2 methods for visualizing DNA in recombinant DNA technology?
Gel electrophoresis
Blotting
What is the process for gel electrophoresis?
DNA passed through gel matrix, which allows certain sizes of DNA or nucleic acids or proteins to pass through it in a size manner
Charged current is passed from negative to positive because DNA is negatively charged
Gel is stained with ethidium bromide (intercalating agent)
Gel put under UV light and DNA bands fluoresce
Probes can locate fragments
- Can stain certain fragments to identify them
Can run DNA, RNA, and proteins through gels
What is the process of blotting and its 3 types?
Gels can be transferred to a solid matrix
- Probe then used to stain molecules on membrane
Types:
- Western – proteins
- Southern – RNA
- Northern – DNA
What are cloning vectors and why is it used in recombinant DNA technology?
Stable, replicating DNA molecule to which a foreign DNA fragment can be attached for introduction into another cell
Putting gene into new source, which allows it to freely replicate and clone itself in new source
Typically plasmid vectors - circular DNA molecules that exist naturally in bacteria
What are the 3 characteristics of a cloning vector?
Origin of replication
Selectable markers – enables any cells containing vector to be identified and selected for
One or more unique restriction site into which the DNA fragment can be inserted
- Fracture vector too heavily if you have more than one restriction site
What are the steps to combining and taking up a cloning vector?
Combining:
- Cut foreign DNA to isolate it, and run it on a gel to confirm/identify it
- Cut plasmid vector at restriction site
- Take foreign DNA sticky ends and join them to the sticky end complements on the plasmid vector DNA
- Sealed by DNA ligase
Taking up:
- Put plasmid vector into medium
- Grow bacteria (likely E. coli) in medium
- Plasmid vector taken up through transformation
- Plasmid vector can freely replicate with foreign DNA
What test is done to know which bacteria will take up a plasmid vector with a gene fragment, and how does it work?
Blue-white screen
Selectable marker is an antibiotic resistant marker that confers antibiotic resistance to E. coli that has taken up plasmid vector
- Any bacteria that doesn’t have plasmid vector (whether it has gene fragment or not) will not grow on blue-white medium
Use gene disruption to determine if gene fragment is present
- Utilize plasmid vector that has a partial lacZ gene (codes beta-galactosidase)
- Restriction site sits inside partial lacZ gene
- If we cut lacZ gene to add gene fragment, the gene will be disrupted
- If lacZ is cut but no fragment is added, it will reseal itself together and will be intact
What are the steps for a blue-white screen?
Grow bacteria on ampicillin and X-gal medium
- Ampicillin selects only antibiotic resistant bacteria (those with selectable marker)
Bacteria with an intact lacZ (lacZ+) gene in plasmid can cleave X-gal
- Produce blue pigment in colonies when they cleave X-gal
Bacteria with disrupted lacZ (lacZ-) cannot cleave X-gal because they don’t have beta-galactosidase
- Produce white pigment in colonies
Pick out white colonies because we only want bacteria with gene fragment in plasmid vector
IGNORE
What are the 4 other types of cloning vectors and when are they used?
Phage-lambda vectors – viral vectors that infect E. coli at a high rate
Cosmids
Bacterial artificial chromosomes (bac) – can hold larger fragments
Expression vectors – replicate gene as well as protein that gene encodes
- Has sequences necessary for both transcription and translation
Used with larger fragments - the larger the fragment, the worse plasmid vectors are
What are the applications of recombinant DNA technology?
Crops/plants
- Genetically modified plants that have herbicide, pesticide, and drought resistance tend to utilize recombinant DNA technology
Vectors that can be utilized to transfer bacterial genes into eukaryotic cells – called yeast artificial chromosomes (yac)
- Can also use retroviral vectors
How is DNA amplified?
Polymerase Chain Reaction (PCR)
Allows for DNA to be exponentially replicated within a few hours
Can do this from very small amounts of DNA
What are the steps of PCR?
Denaturing – DNA is heated up and pulled apart into two single strands
Annealing – primers stuck onto two template strands that specify fragment want to amplify on
Extension – new DNA fragments synthesized by DNA polymerase and dNTPs
- Specific polymerase used is Taq polymerase
Cycle through steps about 30 times to replicate DNA exponentially
What are the 4 limitations to PCR?
Have to have prior knowledge of desired DNA sequence to be able to amplify it
If there’s contamination, the primer may not be specific enough to get the right sequence
Taq is not very accurate because it is fast and lacks proofreading capabilities
- Can alter polymerases to create higher-fidelity polymerases, which decrease error rate by introducing proofreading capabilities
Taq can only replicate small fragments ~2000-2500 base pairs
- Can overcome this by increasing Taq’s fidelity to create longer strands
- Can also modify other reaction conditions to increase the efficiency of Taq
What are the 3 applications of PCR?
Can detect viral DNA in isolates
Can located rare, secretive organisms in an environment
Can detect genetic variation in populations
What is the purpose of sequencing DNA and what are the 5 types?
Determine sequence of bases in a DNA molecule
Types:
- Dideoxy sequencing (Sanger sequencing)
- Sequencing in parallel
- Sequencing by synthesis
- Third generation sequencing
- DNA fingerprinting
What is the basic idea of dideoxy/Sanger sequencing?
Take PCR product (amplified DNA) and use dye terminators (ddNTPs) that lack 3’-OH group that dNTPs have
What is the process for old Sanger sequencing?
Take gene template and do another PCR with ddNTPs instead of regular dNTPs
When ddNTPs get incorporated into new strand, synthesis stops because the ddNTP lacks 3’-OH
Synthesis terminates at different positions on different strands, which generates a set of DNA fragments of various lengths, each ending in a ddNTP with the same base
The fragments produced in each reaction are separated by gel electrophoresis
The sequence can be read directly from the bands that appear on the autoradiograph of the gel, starting from the bottom
The sequence obtained is the complement of the original strand
What is the process for current Sanger sequencing?
Each of the 4 ddNTPs is tagged with a different fluorescent dye, and the Sanger sequencing reaction is carried out
Fragments that end in the same base have the same colored dye attached
The products are denatured, and the DNA fragments produced by the reaction are loaded into a single well on an electrophoresis gel
Fragments migrate through gel according to size and the fluorescent dye on the DNA is detected by a laser beam
Each fragment appears as a peak on the computer printout – color of peak indicates which base is present
What is the limitation of Sanger sequencing?
Since it’s based on PCR, can only get small gene sequences
What is sequencing in parallel?
Millions of DNA fragments sequenced all at once
What is the process for sequencing by synthesis?
DNA is chopped up into small fragments, which end up getting washed across a flow cell in a sequencer, where there are places where fragments can get stuck and attach to flow cell
Primers and polymerases are added to flow cell and the complementary strand of the fragments are synthesized
As synthesis occurs, fluorescent nucleotides are added in and are read by a computer, which reads every nucleotide and what color it fluoresces
After it fluoresces, it is pulled off and a non-fluorescent is added, and then the next fluorescent is added
Can have millions of nucleotides being read on flow cell all at one time
What are the 2 types of third generation sequencing?
PacBio:
- Very accurate and long read lengths
- Typically reads 10,000-200,000 base pairs
- Relatively time consuming
- Tends to be expensive
Oxford nanophore
- Higher error rate, but still long read lengths
- Very small (size of a USB drive), so sequencing can be done in the field
- As DNA passes through nanophore, it disrupts an electrical current in the membrane
- A computer reads the change in current as the base sequence
How does DNA fingerprinting work?
Can identify individuals based on unique genetic markers
Uses microsatellites/short tandem repeats (STRs) found all over the genome
- Number of repeats seen is unique between individuals
Can identify STRs using PCR with primers that sit on either side of STRs
The more STRs, the longer the sequence
Can amplify several STR regions in an individual
- Probability of the same STRs between individuals is very low, especially when amplifying several regions
Homozygotes for an allele will have 1 large peak
Heterozygotes for an allele will have 2 smaller peaks
Can also tell parentage and use in crime scenes because of uniqueness
What is the main form of gene editing used?
CRISPR-Cas9
How does CRISPR-Cas9 work?
Naturally occurring in bacteria and used for immunity and protection against viruses and other invading DNA elements
- CRISPR array consists of palindromic sequence and spacers (sequences from phages or other invaders)
- When a plasmid or phage enters cells, protein cuts up foreign DNA and inserts it into CRISPR array, which is transcribed into precursor RNA that is cleaved into smaller CRISPR-RNA
- CRISPR-RNAs combine with Cas proteins to form effector complexes
- When invading DNA comes in, it finds effector complex and cleaves it to stop it from replicating in host cell
Can be engineered to find unique sequences in the genome for editing
- Can use 2 RNA molecules – CRISPR-RNA and tracr-RNA that combine with CAS9 to form effector complex
- Can then be used to cleave specific DNA sites
- Single guide RNA (sgRNA) can help find genome we want to edit (Seed sequence)
- Can be modified to whatever sequence you want to edit
What are the 2 advantages to CRISPR-Cas9?
Long seed sequences
Applied to many different organisms
What are the 4 disadvantages to CRISPR-Cas9?
Off-site cleavage – cleavage in the wrong place
Can cause genetic mosaics – only alter some cells
Can’t always deliver all components into all cells
A lot of ethics associated
What are the 3 methods of controlling gene function?
Forward genetics
Reverse genetics
RNAi
What is forward genetics?
Begins with a phenotype (a mutant individual) and proceeds to a gene that encodes the phenotype
What is reverse genetics?
Begin with a specific genotype, alter or inhibit it in some way, and determine phenotype from it
Types:
- Site-directed mutagenesis
- Transgenics and knock-outs
What is site-directed mutagenesis?
Cuts out a short sequence of nucleotides with restriction enzymes and then replaces it with a synthetic oligonucleotide that contains the desired mutated sequence
What are transgenics and knockouts?
Transgenics: DNA sequence of interest added to a genome that normally lacks it, then effect of introduced sequence on phenotype is observed
Knockouts: DNA sequence of interest is disabled in a genome and the phenotype is analyzed as it gives a good indication of the function of the missing gene
What is RNAi and why is it used?
Small interfering RNAs and microRNAs can be combined with proteins to form RNA-induced silencing complex (RISC) that cleaves mRNAs and prevents their translation
A way of silencing genes without editing the actual gene
