Biotechnology Flashcards
DNA Technology
Techniques for manipulating DNA
Fundamental processes and concepts to all DNA technology (4):
1) DNA Cloning/Plasmids
2) DNA modifying enzymes (restriction)
3) Gel Electrophoresis
4) Hybridization of complementary nucleic acids
DNA Cloning
The process of isolating a segment of DNA carrying a gene of interest and then making multiple copies of it
–> Allows for the study of specific genes
Plasmids
Small, circular double stranded DNA found in bacteria that are replicated separately from the bacterial chromosome
–> Contain a small # of genes that are helpful but not essential to bacterial survival/reproduction
Plasmids commonly serve as…
Cloning Vectors
Cloning Vectors
A DNA molecule that can carry foreign DNA into a host cell and be replicated there
Formation of Recombinant DNA
The “cutting” and “mixing” of DNA from 2 different sources: Produces recombinant DNA
Recombinant DNA Molecule
Molecule containing DNA from 2 different sources
Gene Cloning
The production of multiple copies of a single gene
Gene cloning has 2 main functions:
1) Gene amplification –> Allows scientists to acquire multiple copies of an isolated gene to then study it in some way
2) Protein Product Formation –> Can transfect bacteria with a gene that encodes for a protein of interest which will then cause the bacteria to produce that proteins in large amounts
Components of a Plasmid Vector
1) Origin of Replication
2) Selective Marker
3) Unique Restriction Sites
4) Gene of Interest
5) Any additional regulatory elements
Origin of Replication in Plasmids
Allows for plasmid replication when added to the chosen host cell
–> If host cell is not bacteria that the plasmid derived from, an additional origin of replication will need to be added that is specifically recognized by the host specie’s replicating mechanisms
Selective Marker
Typically a gene for antibiotic resistance
–> Helps to identify bacteria that have successfully been transfected (taken up the plasmid)
Restriction sites allow for…
The insertion of a gene of interest into a plasmid vector
Additional regulatory elements that can be found in plasmid vectors
Transcriptional promoters, terminators, etc.
–> Adding a promoter makes it so that any gene inserted downstream of it will be transcribed
Process of bacterial transformation with a plasmid
1) Plasmid from bacteria and specific gene from organism of interest are extracted
2) The gene of interest in inserted into plasmid DNA (by restriction) along with a selective marker and any additional elements = recombinant plasmid
3) The recombinant plasmid is then transfected into a bacterial cell
4) The bacteria are placed on an antibiotic to grow: those that grow on it have successfully been transfected
How do we know that a bacteria has been successfully transfected with a plasmid vector?
Through the selective marker which is usually an antibiotic resistance gene:
–> Once transfection has occurred, the bacterial cells are left in an antibiotic solution:
Cells that grow = acquired the resistance gene = successful transfection of plasmid
Cells that die/don’t grow = did not acquire the resistance gene = unsuccessful transfection of plasmid
Restriction Enzymes
Nucleases that recognize SPECIFIC short DNA sequences and CUT the DNA at that DNA site (or very close to it)
Restriction Site
The point at which a restriction enzyme will cut a given DNA
Why is the specificity of restriction enzymes important?
Each restriction enzyme will make the SAME exact cut every time it binds to its recognized sequence
–> Important for the process of recombinant DNA formation as this cleaving in the same manner each time allows for the same “sticky ends” to be created in DNA that are from different sources
Restriction enzymes typically make cuts that produce…
Palindromic fragments –> Same sequence if flipped 180 degrees
How do different restriction enzymes differ?
1) The sequence recognized (restriction site it acts upon)
2) Type of cut made: What type of “ends” the cut produces
Types of cuts produced by restriction enzymes
1) Blunt Ends –> No single stranded overhang
2) “Sticky Ends” –> Single-Stranded Ends: Have a single stranded overhang
Differing DNA molecules cut with the same restriction enzyme will yield…
The exact same “ends”
–> These ends will be complementary to each other and will “find” one another and base pair in solution
Formation of a Plasmid Vector (recombinant DNA process)
1) Plasmid is cut by set of restriction enzymes
2) DNA of interest is isolated and cut out of DNA by same set of restriction enzymes
3) Cut ends of the DNA of interest (DNA to be cloned) and plasmid DNA are “glued” together by ligase
4) Forms recombinant plasmid
Hybridization
Complementary base pairing between 2 single stranded nucleic acid molecules
–> The ability for nucleic acids that are antiparallel AND complementary to “find” each other in solution and connect
Specificity of hybridization allows for…
DNA to be broken apart and then combine together again in the EXACT same way it originally did
Hybridization Probe
Short, synthetic single-stranded DNA fragments designed to base pair with DNA of interest
–> Usually attached to fluorescent or radioactive tags
(typically used for detection and tracking)
What must be known to create a probe?
Must know the sequence of your target DNA to create a complementary probe for it
Gel Electrophoresis
A laboratory method used to separate macromolecules by their rate of movement/migration through a porous gel exposed to an electrical field
Electrodes in the electrical field of a gel
Cathode = At the NEGATIVE end of the gel (electrons flow to it)
Anode = At the POSITIVE end of the gel (electrons flow away from it)
Movement through gel is dependent on…
Size and charge of molecule
What determines how DNA fragments move through a gel? Why?
SOLELY the SIZE of the fragment
–> DNA is negatively charged so it moves towards the positive end of the gel, however since its negative charge is evenly distributed throughout the molecule, the fragments are sorted solely on their size affecting their migration
In a gel, larger molecules _____________ and smaller molecules ____________
Larger Molecules = GREATER friction when moving through gel = Less movement (stays closer to the “top” or beginning point)
Smaller Molecules = LESS friction when moving through gel = MORE movement (goes farther away from start point in gel = closer to the bottom)
How does gel electrophoresis of proteins differ from DNA?
Proteins do not have an evenly distributed charge throughout their molecule usually and so their movement through a gel is dependent on BOTH size and charge
How is gel electrophoresis used to check plasmids?
When a plasmid is made, it can be checked for “correctness” with a gel electrophoresis:
–> The length (in base pairs) of all segments of a plasmid and gene of interest are already known
1) Plasmid is cut through restriction digestion producing both plasmid backbone and gene insert fragments
2) Uncut plasmid, plasmid backbone, and gene insert fragment are all run through a gel
3) Bands for each corresponding component should be found in gel in location that corresponds to BP length expected
3 main biotech techniques:
1) PCR
2) DNA Sequencing
3) CRISPR/Cas
PCR
Polymerase Chain Reaction
Technique for DNA amplification based on DNA replicated reaction
–> Can amplify billions of copies of a target DNA segment in only a few hours
PCR General Procedure
Occurs in 3 step cycles:
1) HEATING = Sample heated to 95C –> Denatures/separates DNA strands
2) COOLING = Sample cooled to 55C –> Allows for the annealing of primers to DNA strands
3) DNA Rep = 72C (temp optimal for DNA rep) – >Allows for DNA polymerase to synthesize new strands
–> Cycle begins again
Materials needed for PCR
1) DNA template (Double stranded DNA with the target sequence in it)
2) dNTPs (Needed for the production of new copies)
3) TWO PRIMERS
4) Buffer
5) Heat Stable DNA Polymerase (Taq Polymerase)
Primers needed for PCR
The primers are made to attach to the 3’ end of the target sequence on both strands of the template
–> Makes it seem as though the primers attach to opposite ends of the separate strands (due to them being antiparallel)
Heat Stable RNA Polymerase
Taq Polymerase
–> Found in hot springs; can withstand the high heat in the first step of the PCR cycle
Full process of PCR:
1) Template DNA mix is HEATED to 95C to denature (separate) the DNA strands
= Strands separate
2) Primers get added while the mix is cooled to 55C = annealing of the primers to the template strands
3) Mix is brought back up to 72C which is optimal for DNA synthesis
= Synthesis of new strands
–> Both the new and old strands then go to be used as templates for the next round of PCR
Newly synthesized strand of PCR is…
SHOTER than the original template since it only consists of the TARGET SEQUENCE (and not the rest of the DNA in the template)
After a certain amount of PCR cycles, the number of molecules produced =
2^n
–> n = # of cycles complete
Applications of PCR (4)
1) Genetic Testing
2) Pathogen Detection
3) Paleogenomics
4) Forensics
Main limitation of PCR
Must have TARGET DNA SEQUENCE info (needs to be known to produce the specific primer!)
DNA sequencing: initial method
“Dideoxy” or Sanger Method
dideoxy nucleotides
Nucleotide with deoxyribose (no OH on 2’C) with the OH group on 3’ C is removed
–> Has no OH on 3’ or 2’ Carbon
Chain Terminator
dideoxy nucleotide
–> Called this because without the free 3’ OH, no nucleotides can be added on causing DNA synthesis to stop
Sanger Sequencing Materials
1) Primer (for synthesis of new strands)
2) dNTPs (Regular: higher conc.)
3) ddNTPs (w/o 3’ C: in LOW conc.)
4) DNA polymerase
5) DNA template
What fragments are produced from Sanger sequencing
Different DNA strands with varying lengths depending on when (random) a ddNTP got added onto the forming chain (and terminated synthesis)
How are the fragments from sanger sequencing analyzed?
Put through gel electrophoresis to sort the fragments by size
How is DNA sequence determined by gel electrophoresis?
Putting the fragments into size order = correct sequence of DNA
Largest fragment = corresponds to 3’ end of the synthesized strand
Smallest fragment = corresponds to 5’ end of the synthesized strand
ddNTPs in DNA sequencing are attached to…
Fluorescent or radio- labels
–> Each ddNTP gets a different tag
CRISPR
Clustered Regularly Interspersed Short Palindromic Repeats
–> First found in bacteria
What is a palindromic sequence?
A sequence of DNA that is read the same 5’ to 3’ on both strand of DNA
Spacers
Fragments of foreign DNA (nestled in between palindromic repeats)
What were spacers evidence of?
Spacers in bacteria were found to be homologous (the same) as sequences in viral DNA
–> Led to discovery of CRISPR/Cas-9 system as an IMMUNE defense against viruses in bacteria
Cas-9
CRISPR associated nuclease –> Cuts DNA
tracrRNA
RNA that bas pairs with palindrome repeat region adjacent to a foreign DNA spacer
–> Involved in effector complex with Cas-9
Effector Complex
Contains:
1) crRNA binded to tracrRNA = sgRNA
2) Cas-9
Guide RNA
sgRNA = crRNA + tracrRNA
–> the crRNA component binds to a specific target within the DNA (spacer) that is complementary to to it
–> “guides” cas-9 to the correct target sequence to be cleaved
PAM Site
5’ —NGG — 3’
–> Any nucleotide followed by GG
–> Binds downstream the target sequence to be cleaved by Cas-9 (cleaving occurs upstream PAM sequence)
What is the role of PAM?
Ensures cas-9 only cleaves the VIRAL/FOREIGN DNA as unless the PAM sequence is found by cas-9, it WILL NOT CLEAVE the DNA
Why won’t CRISPR work in A/T rich regions?
Because they have no PAM sequences (no NGG) –> Cas-9 cleavage would never occur
Double stranded DNA Repair mechanisms
1) NHEJ –> Non-Homologous End Joining
2) HDR –> Homology Directed Repair
Non-Homologous End Joining
Broken DNA ends are directly ligated
–> Usually causes an insertion or deletion at the site in which the ends are reattached
Homology Directed Repair
DNA break is repaired using a donor DNA strand that has regions homologous to the two broken ends
–> In between the homologous regions of the Donor DNA is the sequence that gets copied into the broken region to reattach the ends
dead cas-9
AKA dCas-9
–> Does not cleave the DNA but still binds to it based on the targeting of the guide RNA
–> Can have TFs or fluorescent labels attached to it that can allow for the study of turning genes “on/off” or analyzing chromosome structure
How is cas-9 edited in eukaryotes?
Cas-9 is modified to have a nuclear localization tag for use in eukaryotes
–> Needed because in eukaryotes, the CRISPR/Cas-9 system occurs IN the nucleus whereas in bacteria it only occurs in the cytoplasm
Applications of CRISPR
1) Knockout Creation –> Allows faster production of homozygous recessive mutants as it allows for the simultaneous modification of BOTH loci of a gene
2) Corrective measures –> Has already been used to successfully correct defects that are linked to diseases encoded by ONE gene
3) GMOs
2 main problems with CRISPR
1) Potential “off-target” cleaving
2) Ethical Concerns
“Off-target” cleaving
The possibility that a synthesized guide RNA (sgRNA) will target an unknown gene rather than the one intended which can cause cleavage at the wrong site
Current agreement to ethical concerns of CRISPR
CRISPR can only be used in humans to make edits that CANNOT BE INHERITED
Genomic Library
Represents a collection of cloned DNA fragments derived from the entire genome of a source organism
–> Contains ALL parts of the genome (non-coding AND coding)
How are DNA libraries stored?
Stored in a population of vectors, each containing inserts/fragments of DNA from the genome
–> These vectors are then stored in bacterial colonies usually
Genomic vs cDNA Library Contents
Genomic Library = ALL of the genome (Introns, Exons, Promoters, Enhancers)
cDNA Library = Only contains the DNA that gets transcribed into mRNA (the protein encoding DNA)
–> EXONS ONLY
Creating of a Genomic Library Process
1) Take double stranded DNA of the genome and conduct random restriction digestion
2) Fragments produced from restriction cleavage are then inserted into plasmids
3) Bacteria are transfected with recombinant plasmids (Each bacterial cell takes in ONE plasmid)
4) Colonies of bacteria form: Each colony represents identical bacteria with the same plasmid containing DNA fragment
= Genomic Library
Random Restriction Digestion
Throughout the DNA, restriction sites are randomly naturally occurring so to break up the genome for creation of a library, these random restriction sites are utilized
cDNA
Complementary DNA
–> DNA produced from mRNA as the template
What enzyme is needed for cDNA?
Reverse Transcriptase
Process for producing cDNA
In the nucleus:
1) DNA undergoes transcription to produce pre-mRNA
2) Pre-mRNA is spliced to produce mature mRNA with JUST EXONS
3) Mature mRNA is isolated from the nucleus
In Test Tube:
4) Isolated mRNA is added to a test tube containing Reverse Transcriptase
5) A single cDNA strand is produced from the mRNA template using reverse transcriptase
6) A second strand of cDNA is formed using the first cDNA strand as the template
–> The double stranded cDNA can then undergo similar process as genomic library formation to produce a cDNA library
cDNA Library
A pooled collection of all genes that are actually transcribed into proteins
–> All the protein encoding DNA
–> A more limited library; doesn’t represent entire genome
Advantages of cDNA Library
1) Allows focus of study on genes responsible for specialized functions
–> Analysis of different cell types or developmental stages
2) Gets around the problem of introns
