Topic 3 Flashcards
Restriction Endonucleases
- Enzymes
- cleave double stranded DNA
- at restriction site (specific sequences)
4 Steps to Clone DNA using Restriction Endonucleases
Step 1
- Produce DNA fragments - digestion with restriction enzymes. Ligate fragments with appropriate vectors (plasmids).
4 Steps to Clone DNA using Restriction Endonucleases
Step 2
- Insert DNA fragment into a cloning vector (plasmid). i.e. DNA fragment A + plasmid = recombinant plasmid A, DNA frag B + plasmid = recombinant plasmid B. Transform plasmids into (bacteria) E.coli to amplify the cloned DNA.
4 Steps to Clone DNA using Restriction Endonucleases
Step 3
- Plate cells on agar plate. Colony of identical cells. Select colony with plasmid of interest.
4 Steps to Clone DNA using Restriction Endonucleases
Step 4
- Amplify plasmid DNA. Grow E.coli cell with plasmid in broth culture to amplify recombinant plasmid DNA. Isolate DNA by lysing the bacteria E.coli cell. Analyze plasmid DNA
DNA cut by restriction enzymes
- have single stranded overhangs
- ends are cohesive (complementary, sticky) - re-anneal/ligate
Function of restriction enzymes in bacteria.
Foreign DNA:
-protect genetic material of bacteria from foreign DNA (virus)
-restrict foreign intrusion which leads to cut up DNA
Bacterial genome:
- protected from RE degradation by methylation of nucleotides within sequence (methylation of bacterial genome)
-RE recognizes/blocked by methyl groups.
Restriction enzymes recognize ______ sequences
Palindromic.
-staggered, or blunt ends.
(DNA is identical but inverted in the complementary strand.
Cloning plasmids (Plasmid cloning vectors)
- ds extrachromosomal circular DNA
- replicate independent of cell chromosome (therefore, many plasmids/cell)
3 essential components to Cloning Plasmids
- origin of DNA replication (for amplification in bacterial cells)
- selectable marker (ab resistance gene)
- unique RE cleaving site; outside origin, resistance gene i.e. MCS - this is where you insert foreign DNA of interest
Expression plasmids
- designed for expression of proteins in bacteria, plants, yeast, or animal cells
- must be able to replicate in bacteria –> to amplify plasmid
- contain selectable marker to identify the bacteria with plasmid (vs bacteria without plasmid)
- promoter to express the cloned gene (inserted at MCS - the encoded protein)
- translational regulatory elements (polyA signal
Reporter plasmid
- MCS where you insert gene regulatory sequence (i.e. promoter)
- MCS upstream of reporter gene
- reporter gene encodes protein that can be visualized
- origin of replication
- no promoter
DNA and RNA electrophoresis
- separate mixture of DNA/RNA fragments
- in porous gel matrix (agarose/polyacrylamide) in electric field
- black to red (- to +) DNA is -
- inversely proportional to size; large move slower, short move faster
- separation is based on size, not molecular mass
Southern Blot Procedure
- digest DNA with restriction enzyme
- gel electrophoresis to separate molecules
- transfer and immobilize DNA fragments from electrophoretic gel onto DNA-binding membrane.
- denature DNA
- molecular hybridization radioactive probe DNA to membrane
- detect presence of specific DNA fragments X ray film
Molecular Hybridization
annealing of DNA strand with complementary DNA strand
- prior denaturation (separate 2 strands of DNA)
- hybridization: renaturing when temperature reduced
i. e. hybridize (anneal) radioactive probe to complementary strand of denatured target DNA
Transfer DNA from gel to DNA binding membrane (Southern blot)
- glass plate (support)
- agarose gel with DNA in transfer solution buffer
- transfer solution on Whatman sheets act as wicks
- nylon membrane where DNA is immobilized
Northern Blot
-mRNA not RE digested DNA
similar procedure to southern blot
-transfer size fractioned RNA from electrophoresis gel to membrane support
Applications of Northern Blot (purpose)
- to detect length and types of transcripts from specific gene
- quantitative measurement of transcription level of gene
- measure changes in gene expression as a result of developmental stages, environmental stress, drug etc… i.e. heat shock gene in embryos
Polymerase chain reaction (PCR)
-synthetic nucleotides complementary to known sequences used to prime enzymatic amplification of the sequence of interest
-amplification occurs exponentially; each cycle doubles number of molecules of sequence of interest
Cycles:copies = linear relationship, exponential growth
3 steps of PCR
- denature of genomic DNA (95C)
- anneal of denatured ssDNA to oligonucleotide primers (50-60)
- Primer DNA extension: replication of DNA segment between sites complementary to primers from provided 3’OH (70-72)
Repeat cycle 20-30 times
Why is DNA polymerase not ideal for PCR?
DNA polymerase is not synthesized at 37 degrees.
It is not heat stable.
What are the two thermostable polymerases?
Taq polymerase (used for PCR) and Pfu polymerase.
Taq polymerase
- thermostable DNA polymerase (above 90C)
- best choice for PCR
- lacks proofreading activity, so errors introduced into amplified DNA are low but significant frequencies (low fidelity)
- does not amplify fragments larger than few thousand base pairs well
Pfu polymerase
- thermostable DNA polymerase
- very low error rate (high fidelity)
- best choice for gene cloning
Tfl
- thermostable DNA polymerase
- high fidelity
- long segments of DNA (35kb)
Applications of PCR
- gene cloning
- gene diagnostic
- DNA quantification
- measure gene expression (RT reverse transcriptase mediated PCR)
- site-directed mutagenesis
Gene Cloning
- must have gene sequence to design the primers
- use Pfu cause you want high fidelity and PCR requires high temp
- create plasmid to express HGH cDNA fused to GFP – insert at MCS; use PCR to clone
How to design primers
- add restriction sites
- primer with restriction site to generate sticky ends and insert into the DNA
- the restriction site of the primer will not attach, but that will be the cleavage site
- everything in between (ORF) will be cloned because the primers will bind
i. e. HindIII –> EcoRI
Gene diagnostic
- prenatal diagnosis of inherited human diseases
- forensic identification using small amounts of biological samples
Examples of uses of PCR for gene diagnostic
- Huntington’s
- sickle cell
- DNA profiling (STR: short tandem repeats, CODIS)
- paternity tests
- forensics
DNA quantification
- measure abundance of microbe in sample
i. e. Quantitative real time PCR = qPCR
Quantitative real time PCR (qPCR)
- measures the abundance of microbe in the sample via accumulation of fluorescence
1. fluorescent dye incorporated into amplified DNA
2. amount of fluorescent DNA increases with each PCR cycle
3. fluorescence can be measured at each PCR cycle
4. samples with more starting material (target DNA) will produce fluorescent signal that crosses threshold earlier than less abundant DNA sample
Measure gene expression by RT-PCR (reverse transcriptase)
- copies mRNA to cDNA
- amplify and quantify
- detect and quantify amount of specific mRNA in different samples
- clone gene of interest by creating and amplifying cDNA
Steps to RT PCR
- isolate mRNA
- reverse transcribe single strand mRNA to ss cDNA (using olig-dT or gene specific primers and viral reverse transcriptase
- PCR amplification of cDNA to dsDNA (with forward and reverse primers; one primer/strand cuz single strand) - extension
- compare quantity of PCR like qPCR
Analysis of proteins via SDS-PAGE
- used to separate all polypeptides
- protein bands in gel
- visualized coomassie blue staining
Analysis of proteins via Western Blotting
-what can it be used for?
aka immunoblotting
- used to compare protein abundance in different cell types (normal vs cancer)
- transfer of separated proteins from gel to solid support (membrane)
- detection of target protein with specific antibody
SDS page steps
- cells lysed in buffer (denature) by detergent sodium dodeycl sulfate
- SDS binds proteins giving negative charge, migrates to positive
- polypeptides move through polyacrylamide gel through porous polyacrylamide gel matrix in electric field (electrophoresis)
- mobility is linear to molecular mass (smaller move faster tahn larger)
Western Blot procedure
- transfer apparatus
- negative charged proteins move towards positive electrode and trapped on blotting membrane (upwards)
- membrane incubated with blocking solution to prevent non-specific ab to bind - detection
- membrane with protein bands incubated with antibody
- antibody will bind to transferred protein only if it is specific
- membrane bound antibodies detected via radioactivity, colourimetric, fluorescence
Immunofluorescence imaging
-protein localization
Steps for immunofluorescence imaging
- grow cells on a glass slide
- fix cells with paraformaldehyde
- block with protein solution
- incubate with 1 antibody (detect protein of interest)
- wash off unbound antibody
- incubate with 2 antibody (bind 1 antibody)
- wash
- analyze with fluorescence microscope
GFP
green fluorescent protein
- fused to ORF to determine localization of protein
- protein tag
- tagging on N and C terminus will not allow for expression
- must be localized in frame with the ORF in order to view in real time by UV fluorescence
- image tells you how protein moves in the cell
