BIOMG 3320 LEC 23-25

What does cloning DNA into plasmids require?

restriction enzymes and DNA ligase

What do restriction enzymes do?

They cut dsDNA at specific, short palindromic sequence and create "sticky ends"

What allows DNA to be ligated together and why?

DNA cut with the same restriction enzyme can be ligated together bc of overlapping sticky ends

What are vectors?

Plasmids are vectors

What are plasmids?

Plasmids are circular DNA molecules that are separate from the bacterial genomic DNA

What do plasmids contain and what does this allow them to do?

Plasmids contain their own origins of replication and therefore can replicate independently from the genomic DNA

What do plasmids cary

They carry antibiotic resistance genes

How many bp of DNA does plasmids allow cloning of ?

Up to 15000 bp. It can insert a piece of your DNA of choice up to 15000 bp into the plasmid

What is a cloning vectors purpose

To amplify a gene of interest and make lots of DNA

What must cloning vectors contain?

Cloning/restriction enzyme site that can be cleaved to insert your DNA of choice (that has been cut with the same restriction enzyme) || Origin of replication (plasmid cannot replicate without it) || Selection marker (such as antibiotic resistance)

What is an expression vector's purpose?

To amplify protein product of gene of interest in order to make a lot of protein

What must expression vectors contain?

cloning/restriction enzyme site || Pigeon of replication || Selection marker || Protein expression cassette (i.e Operator, Promoter) || Transcription Terminator Sequence || Ribosome binding site || Gene encoding repressor that binds O and regulates P

What are the cloning procedures

1. Cleave/digest vector and DNA fragment with proper DNA restriction enzymes || 2. Ligate the digested vector with the DNA fragment || 3. Transform the recombined plasmid into E. Coli cells (transformation) || 4. Grow up bacterial colonies on plates continuing desired antibiotics (your vector will be resistant to chosen antibiotic) || 5. Select the right clone that contains your targeted DNA

What are the materials required for Polymerase Chain Reaction?

Template DNA || 5' primer || 3' primer || 4 types of dNTP || Taq polymerase

What is the procedure for PCR

Put all materials together in a tube || Cycle through the following temperature changes || 90 deg: separates dsDNA to ssDNA || 55 deg: anneals (ssDNA goes back to dsDNA) || 72 deg: elongation (Taq enzyme works best at this temperature)

Describe how to do primer design

1. Determine sequence of complementary strand if given single strand of DNA to make primers for || 2. Make short sequences of DNA complementary to the 3' end of the template and the complementary strand || Because DNA synthesis proceeds in 5' to 3' direction, the primer must bind to the 3' end of DNA and proceed in the 5' to 3' direction || 3. You will have a 5' primer and 3' primer || 4. The 5' primer is the same as the 5' end of the template (complementary to the 3' end of other strand) || The 3' primer is complementary to the 3' end of the template strand

What are some adaptations to PCR

1. Reverse transcriptase PCR (RT-PCR)

Describe reverse transcriptase PCR

1. Start with RNA instead of DNA || 2. Use reverse transcriptase to convert RNA to DNA || 3. Following steps are the same as regular PCR

What is the purpose of mRNA polyA tails

Helps in purification of mRNA and serves as a universal template

What is used to synthesize DNA strands in cDNA libraries

mRNA is used as a template and is catalyzed by reverse transcriptase

What is duplex DNA

cDNA

How do you construct a cDNA library?

1. Purify mRNA || 2. Oligo-T (TTTTTT) primer anneals to polyA tail of mRNA || 3. Reverse transcriptase creates mRNA-DNA hybrid || 4. mRNA is degraded with alkali || 5. To prime synthesis of second strand, generate oligonucleotide of known sequence to ligate to 3' end of the cDNA || 6. Normal amplification steps follow: DNA pol 1 and dNTPds to extend primers to make dsDNA

What are common elements in cloning and expression vectors?

Origin of replication || Antibiotic resistance genes || Restriction enzyme cutting sites

What are unique features of expression vectors

promoter/operator || Ribosomal binding sequence (S-D sequence) || Transcriptional termination sequence || Genes encoding the repressor

What does site directed mutagenesis allow for?

Allows researchers to individually change specific amino acids in a protein to see how it alters function

What is the method for site directed mutagenesis

1. Start with a plasmid that contains gnee and target site for mutation || 2. This reaction takes place in a tube (not within bacterial cells) || 3. Design primers with mutation of choice to target mutation site || Alter DNA nucleotide so that amino acid of choice will be changed || Make 2 primers which each contain the mutation || 4. Use DNA polymerase to extend and incorporate the mutagenic primers (a single base pair mutation will not prevent annealing of primers || 5. Several cycles of PCR later, you will have several plasmids with mutation incorporated || 6. Digest nonmutated parental DNA template with methylation specific nuclease and anneal newly synthesized strand || 7. You are left with mutated plasmid with nicked stand || 8. Transform dsDNA into cells. Cells repair nick in mutated plasmid

What are fluorescent fusion stages for detecting protein in vivo localization

You can mutate GFP to make different fluorescently colored proteins || Adding GFP to a protein allows you to visualize that proteins location within a cell/tissue under a microscope

How do you fuse GFP to protein of interest

Fuse the GFP to your gene of interest so that the DNA will be transcribed together and then translated into a single chimeric protein || For the gene, use cDNA that contains only exons

What can fluorescence be used for

To determine protein localization in vivo

What are Techniques to identify protein-protein interaction

Coimmunoprecipitation || Epitope tags for comimmunoprecipitation || Yeast-two hybrid

What is co immunoprecipitation

1. Conjugate antibody against your protein of interest to a bead || 2. Incubate bead bound antibodies to your sample. The antibody will bind your protein of interest || 3. Wash away excess || 4. Spin down to pull beads down. Identify what other proteins were pulled down with your protein. This tells you that your protein of interest was interacting with this other protein

What are epitope tags for co immunoprecipitation

Used when you have an antibody against your protein of interest? || 1. Fuse a known epitope (small protein) to your protein of interest || 2. Pull down your protein using antibodies against the known epitope || All proteins isolated can be separated and identified

What is the yeast two hybrid?

In yeast two hybrid analysis, the protein coding regions of the genes to be analyzed are fused to the yeast gene for either the DNA binding domain or the activation domain of Gal4p, and the resulting genes express a series of fusion proteins

What is the example used for yeast two hybrid analysis?

1. Fuse Gene of interest, X, to the activating domain in yeast strain 1. || 2. Fuse gene of interest, Y, to the DNA binding Domain in yeast strain 2 || 3. Mate yeast strain 1 and yeast strain 2. || 4. If protein X and Y interact, they will bring together the DNA binding and activating domains and the gene will be transcribed. || 5. Plate the yeast in a medium that requires interaction of the binding and activation domain for cell survival || 6. Sequence fusion proteins to see which proteins are interacting

What is the difference between co immunoprecipitation and epitope tags for co immunoprecipitation

How are each of these similar vs different

What has recombinant DNA technology been used for

To alter genomes for the purpose of treating diseases, producing medicine and crops, and drive research

What can DNA homologous recombination do?

Homologous regions between two DNA strands can pair and swap to produce recombinant chromosomes || It can also be used to delete genes (knockouts) or modify genes (transgenic variants)

What type of research is homologous recombination common in and why

In yeast research. Yeast have 6000 genes and are a simple eukaryotic model organism

How do you alter yeast genomes via homologous recombination?

1. Design a DNA fragment to encode a resistance gene flanked by regions homologous to the regions flanking your gene of interest || 2. Introduce the artificial designed DNA into yeast || 3. Yeast will integrate the DNA fragment into their genome via HR || 4. The knockout/mutant strain of yeast can be selected via the selectable marker

Is creating knockouts/mutans in yeast easy

Yes, it only takes a few weeks

Is it easy to create knockouts/mutans in mice and other mammals

No, it is much more difficult

Why is conventional mutagenesis via homologous recombination difficult in mice

1. Complex DNA design || 2. Difficult selection process || 3. Generations of breeding (several months) to get desired transgenic mice

What system has replaced conventional mutagens and became the current go to method for genome editing?

Crispr/cas9

What does CRISPR and CAS stand for

Clustered regularly interspaced short palindromic repeats || CRISPR-associated proteins

How was the CRISPR-Cas system discovered and adopted for genome editing?

Mojical studied an archaeal microbe (Haloferax meterranei) and found that there are 30 bp interspaced identical palindromic sequences with 36 bp unique spacer sequences between them || 2. Similar sequences were reported in E.coli strain K122 || 3. The variable spacer sequences were homologous to viral genomes or external plasmids, so it was hypothesized that CRISPR was an immunity mechanism that protects transmissible genomic elements

What was CRISPR hypothesized as

An immunity mechanism that protects transmissible genomic elements

How does the CRISPR/Cas9 system work

1. Bacteria genomes have CRISPR arrays, Cas genes, and tracrRNAgenes || 2. tracrRNA is transcribed front he tracrRNA gene and the precrRNA is transcribed from the CRISPR Array || 3. Pre-crRNA is processed into pieces containing one palindromic repeat and one spacer each(these pieces are called crRNAs) || 4. Palindromic sequences within crRNAs can base pair with tracrRNA and form a functional complex with Cas9 || 5. Once foreign DNA is introduced into the cell, the spacer sequence will base pair with the foreign DNA (DNA-RNA duplex has higher binding affinity than DNA-DNA duplex, so the foreign DNA will preferentially bind to the spacer sequence within crRNA) || 6.) After the spacer sequence base pairs with the foreign DNA, Cas9 endonuclease activity is activated, and Cas9 cuts the foreign DNA

How is CRISPR/Cas9 used for genome editing?

sgRNA combines the functions of tracrRNA and crRNA into one molecule with the unique spacer sequence engineered to match teh gene of interest || 2. sgRNA and Cas9 can be cloned into a single plasmid || 3. The plasmid encoding sgRNA and Cas9 is introduced into cells, where sgRNA and Cas9 assemble into a functional complex || 4. The functional sgRNA/Cas9 complex scans the genome to base pair with its target gene || 5. Cas9 nuclease makes a double stranded break at the target site, and non-homologous end joining (NHEJ) will repair the break and introduce a mutation in the process

What can you use to introduce a specific mutation instead of a random one?

Use a mutated Cas9 with one inactivated nuclease site, which will only nick a single strand

How do you introduce a mutation of your choosing

A recombinant dsDNA fragment that include the desired mutation must also be delivered to the cells-this donor dsDNA will get incorporated into the nicked gene via homologous recombination

What can modified Cas9 be further engineered for

Transcriptional repression by coupling Cas9 to a transcriptional repressor and deactivating the nuclease function

What are advantages of CRISPR Cas systems over conventional mutagenesis

Simple design of sgRNA/donor DNA || CRISPR/CAS9 plasmids can be directly injected into zygotes || Order of magnitude more efficient than conventional mutagenesis || Additional capabilities aside from genome edition

Does CRISPR Cas9 complex target specific DNA sites by DNA binding motifs encoded in the Cas 9 protein

No

Is CRISPR/Cas system a bacterial immune system defending attacks by bacteriophages

Yes

Does cleavage of DNA double strands by Cas9 often resulted in imprecise NHEJ repair and gene inactivation

Yes

Can CRISPR/Cas9 be engineered to regulate gene expression with DNA cleavage

Yes