How do you do somatic cell nuclear transfer?
- took nucleus from mammary/adult cell of sheep
- took oocyte from another cell and took nucleus out and fused other cell with this cell.
- Zapped it to cause a membrane potential that initiates cleavages of this embryo which dev into Dolly
Whats a clone?
an exact genetic copy: bacteria are clones since they reproduce by binary fission. Dolly the sheep is a clone of her mother.
what’s recombinant DNA?
Cloning a single fragment of a gene: Joining of a DNA seq of interest to DNA orginating from another source. This allows the isolation and amplification of a specific region of chromosomal DNA. Once amplified, detailed analyses of the DNA structure/protein function are possible.
Complementary ____ pairing is the basis for most recombinant techniques
what are cloning tools
- source of DNA with gene of interest
- vector (our prokaryotic/low form of DNA)
- sev bacterial enzymes & processes
- selection methods: to identify bacterial hosts harboring recombinant vectors. To identify clone with specific DNA of interest.
How do you produce a library of clones
- extract sample DNA (complementary DNA; comp to mRNA), genomic, chromosome specific)
- cut sample DNA and vector DNA with same restriction enzyme: plasmid, phage, cosmid or YAC
- Mix, and ligate
- Transform/transfect into host: transformation/transduction
- plate on selective media
- make replicate
- screen for specific DNA fragment.
what are the sources of human DNA containing the gene you wish to clone?
- whole genomic DNA
- chromosome specific DNA
- cDNA/tissue specific cDNA
how do you make cDNA?
Using a retroviral enzyme , reverse transcriptase to make DNA copies of mRNA. Starting material is mRNA isolated from a tissue/organism which has polyadenylated tail added to it which is exploited by making poly T fragment that pairs with it which forms a primer for DNA polymerase. Then add reverse transcriptase. DNA polymerase cant use RNA as a template but Reverse transcriptase does (from retroviruses) which makes a DNA copy! Get rid of mRNA by using alkali and leaves DNA by itself and make a second DNA copy by making DNa pol 1.
what are vectors
DNA molecules that can replicate autonomously in a host:
- plasmids: <5-10kb
- bacteriophage: 20kb
- cosmids: 50 kb
- yeast artificial chromosomes; linear, eukaryotic; 100-1000kb
what are features of pBR322 plasmid?
- R genes
- RE sites:
-PstI: amp R marker
EcoRI, BamHI, SalI : Inactives tetracyclin
unique endoclease cut sites which linearizes it and makes sticky ends which allows u to put piece of DNA in there and recircularize it.
ORI: replicates itself autonomously which makes lots of copies of DNa inserted in there
Bacteriophage have 2 choices to infect bacteria:
- Lytic: makes lots of copies of themselves and make new protein capsids and lyse cell and kill host.
- Lysogenic cycle: results in production of “dormant” prophage. We dont want this! We want it to replicate
what do we do if bacteriophage goes into lysogenic cycle?
stuffer piece: houses the genes req for lysogeny. WE cut these out and replace with human DNa which forces it go to lytic cycle.
what are features of yeast artificual chromosomes
- RE sites: EcoRI, BamHI,
- antibiotic selectable markers
what are features of Type II restriction endonucleases?
- bacterial defense mechanism
- DS DNA seq specificity; rec 2-8 bp palindromes (read same way backwards and forwards)
- many produce sticky ends
- DNA ligase: seals the gap
Recombinant DNA is transferred into bacterial host: Used by plasmids; Bacteria take up recombinnat plasmids and start replicating inside bacterium. Treat E. coli with CaCl2 and cold to take up DNA. Mix recombinant plasmids with bacteria and plate transformed bacteria onto agar.
What is the process for bacteriophages?
Transduction: Infect bacteria with recombinant virus. Take out stuffer pieces and mix phage construct with bacteria in agar. Pour into plates
After plating plasmids + bacteria how do we decipher which colonies to choose?
The one that has foreign DNA. So for cells that grow on tetracycline but not on tet + amp contain recombinant plasmids with disrupted amp Resistance. , hence the foreign DNA, hence the one we’re going to use.
How does phage cloning work?
Genes for viral integration into host genome (lysogenic life cycle) are removed and replaced by foreign DNA. Take out stuffer piece and insert foreign DNa, only certain ones get replicated. Make it into bacteriophage and infect bacteria onto soft agar. The bacteria grow within agar, so agar constrains bacteria when it lyses. The bacteriophage goes into bacteria, make lots of copies and lyse the bacteria. If on top of media it can go to far. This makes plaques, that are clearings where bacteriophage have lysed those cells. Each clearing holds a lot of bacteriophage. Their all clones of each other.
Only recombinant phage are infective and will form ____
How do Yeast artificial chromosomes work?
- Linearize and put piece of DNA.
2. Digest away cell wall so it could take away DNA from envmt to make yeast cell.
what are some commonly used probes
- coned DNA fragment from another species
- Anitbody specific for gene product
- PCR amplified DNA fragment
- synthesized DNA fragment based on protein sequence.
Probes are used in _____reactions and must have a high degree of bp complementarity to the gene of interest. The pattern of colonies is transferred to a membrane; bacteria/phage are lysed; DNA is denatured by exposure to strong base (NaOH). Labeled probe is heat denatured; membranes are bathed in a solution conducive to comp base pairing bet the probe and immobilized DNA. Membranes are washed, dried, and dev to detect regions of probe/DNA hybridization.
What can you do after you have a clone?
- Seq it: DNA seq det. protein seq, so many clues about protein function can be derived from the DNA seq of its gene.
- Express it: Recomb proteins can by synthesized (how we get insulin, human GH)
- Use it as a probe: use to detect mutations in genes. Use cloned minisatellite seq as probes to prod DNA fingerprints for ind identification
- Use it in functional studies: detect mRNA exp of the gene in tissues, ind cells, in response to sig molecules
- transfer it: gene therapy correct a genetic defect.
What are applications of cloned DNA sequences
- sequence analysis
- molecular probes: southern blots: forensics/patient diagnosis
northern blots: gene exp studies
in situ hybridization
- analysis/production of gene product.
How is DNA sequencing done via the Sanger Dideoxy Method (ddNTP)
ddNTP analogs inhibit DNA pol as it synthesizes the complementary strand.
- 4 rx mixes are set up; each has a diff ddNTP analog + 35S labeled dNTPs
- ddNTP analog is added in a specific low ddNTP: dNTP ratio
- since dNTPs»_space; ddNTPs, synthesis will terminate at various points in seq.
- each mixture is sep by PAGE
How does the automated DNA sequencing work?
Start with fragments thats labeled with flourescence probe. Still adding ddNTP analogs that halts replication at various point along seq of DNA. Now they all have diff label and we can mix them all together and throw on PAGe and will be seq via a detector on comp screen.
how you do you analyze/synthesize a gene product?
once isolated, a cloned gene can be excised from the cloning vector and transferred to a diff, more specialized vector which is called SUBCLONING.
What can subclones be used for?
- site directed mutagenesis (where you int mutation in seq of gene, if you change aa seq in domain and you see it lose function it tells you where active site of enzyme is)
- fusion protein production: can follow protein around
- in vitro translation: to protein product
- production of recombinant proteins.
what is site directed mutagenesis? how does it work?
Used to map functional domains of the gene product: Used to do functional studies on protein product.
what are expression vectors
specialized plasmids are used to express proteins in bacteria. This is where we get insulin, hGH, etc. If you want bacteria to make protein for you. Has ribosome binding site. (shine delgarno seq). Have promotors (bind activating TF’s) and operators ( bind repressors)
What are fusion proteins?
GFP: Used to tag proteins to monitor when and where they are expressed in an org
How does Southern blotting work.
Take RE digested DNA (whole genomic DNA) sep by agarose gel electrophoresis (bigger pores) and will sep them based on size. the pattern of Dna fragments transferred to solid support so you can expose piece of paper to probe. Take filter paper and soak to NaOH to make DNA ss so it could comp pair with labeled piece of DNA so it could use it as a probe.
Whats diff bet. Northern and southern blotting.
Have mRNAs instead and you can see whether those patterns are being changed.
What is Southern blotting used for?
forensic analysis. Repetitive element probes are used to identify ind patterns.
WHat is RFLP analysis (restriction fragment length polymorphism)
Diff alleles can have diff DNA seq on chromsomes which are called polymorphisms. RFLP occurs when a polymorphism either creates a new restriction enzyme site or ablates an existing site. You want a DNA probe that overlaps this restriction enzyme site and will give you 2 pieces or 1 big piece. Change results in variations in DNA patterns identified by Southern blotting.
What is RFLP analysis used for.
Prenatal diagnosis. Uses the concept of linkage.
what is microarray technology?
useful for comparing genes expressed in 2 populations of cells or tissues.
Uses mRNA! 2 dev stages of frog. They take all mRNA from early and later stage and make complementary DNA from mRNA using florescently labeled dNNTP, mix them together and expose them to membrane. Each spot rep’s a complementary seq DNA specific for a specific gene. You look for color. You can see which mRNA or which proteins are being expressed at the two diff developmental stages.
What is amplichip CYP450?
CYP2D6 CYP2C19: cytochrome P proteins; liver metabolizes drugs and hydroxylates them so we could excrete them. Some drugs have to be metabolized in active form/ some have to be metabolized to be excreted.. Categorizes patients as ultra rapid, extensive, intermediate or poor metabolizers. helps det which drugs will be most effective for that person.
what are array technologies used for
Prognosis markers for some types of cancers. Reveals the prognostic indicators of sets of proteins being exp in your specific tumor. Have arrays based on breast cancer recurrence risk, or metastatsis risk, prostate caner, lung caner prognosis or colon cancer.
siRNA is useful for:
useful for knocking down protein levels and monitoring effect on cell behavior.
what is PCR
Makes multiple copies from a very small amt of starting material. Allows the sel amplification of a single molecule of DNA sev millionfold in just a few hours. Must know DNA seq in region surr DNA to be amplified. Human genome project will support use of this technique for gene cloning.
Standard variable nucleotide random repeat requires ____ng of DNA. PCR req 0.2 ng which can be obtained from
500; saliva on cig butt or a 1mm blood stain.
what are the steps of PCR
- heat denature
- anneal primers
- DNA pol (taq) makes copies in both directions.
log inc in numner of copies in each cycle. 2. 4. 8
PCR amplified exons can be sequenced for _____.
What is ARMS/Allele specific PCR
Used to detect point mutations using allele specific primers.
whats the chart to deceipher how strong a disease is?
would use allele specific PCR:
- Class 1: strong evidence of genetic disease. Absent from control population. Genetic testing of all first deg relatives is recommended.
- Class 2: mutation may be disease causing or benign. Typically absent from control population.
- Class 3: not expected to be disease causing. Typically common in control pop. Genetic testing is not advised.
Combined DNA Index System: use microsatellite probes. Use 13 DNA regions = 1 in 10^9 probability of identity.
What are some challenges of transferring genes into animal cells
- vectors must contain eukaryotic promoter elements (transgene)
- gene must be integrated into the host chromosome to allow passage to the next generation.
- Site of integration is mostly random.
How do we make transgenic animals
- microinjection of DNA directly into a fertilized egg cell
- careful selection/breeding leads to new strains harboring the introduced gene.
- If we introduce inactive gene and homologously recombine it into the corre site in genome you get a KNOCKOUT which is useful in dev studies.
____is the term used for introducing genes for human proteins or pharmaceuticals into traditional farm animals and harvesting the products (throug their milk etc)
whats gene therapy. What’s ex vivo?
take out cells that are most affected, manipulate it and reintroduce it. Take bone marrow stem cells. Currently the most reliable form.
whats in vivo gene therapy?
corrected gene is injected into patient. more controversial
what are non viral delivery systems for gene delivery
- non lipid coats
- naked DNA
- mini chromosomes
what are current targets for gene therapy
- Cystic fibrosis
- muscular dystrophy
- huntingtons chorea
Currently, it is illegal to perform gene therapy on: ____cells or _____
what are strategies for cancer gene therapy
- suicide genes;
- anti angiogenesis
- tumor suppressor genes
____ is disease targeted for gene therapy. How does it work?
choroideremia. X linked disease; escort protein for G protein called Rab that needs to get prenylated in order to stick in cell membrane. If this doesnt happen it causes degeneration of retina which can lead to blindness. Confined to the eye. Introduce the gene using a cannula which adds fluid to lift retina and insert DNA there.
How is gene therapy involved in dentistry
- bone regeneration
- periodontal ligament regeneration
- dentin regeneration
- salivary gland regeneration
- oral cancer treatment
how do stem cells work?
take cells from blastocyst (from IVF embryos) –> pluripotent stem cells–> make lots of diff kinds of tissues.
what are the types of stem cells?
Totipotent: from zygote and can form whole human
Pluripotent: from blastocyst and can form cell types from all 3 germ layers.
Multipotent: from adult. Can form many closely related cell types.
Major diff bet embryonic and adult stem cells is that embryonic are ___ wherea, adult cells are multipotent. Embryonic stem cells are easy to identify, maintain, isolate, and grow. Adult stem cells are not. ____ stem cells are not found in all tissues. ____ stem cells have a risk of rejection and there is an ethical dilema. No risk of rejection for adult stem cells.
pluripotent; Adult; Embryonic
What are future directions for stem cells?
- somatic cell nuclear transfer: transfer adult cell nucleus into enucleated egg, stimulate to dev into blastocyst and harvest embryonic stem cells. (not succesful in human cells)
- iPS cells: Induced pluripotent stem cells: introduce 4 TF genes into adult diff cells. Induces them to reprogram into stem cells. Not yet succ in human cells.