Flashcards in DNA techniques Deck (62)
How do you do somatic cell nuclear transfer?
1. took nucleus from mammary/adult cell of sheep
2. took oocyte from another cell and took nucleus out and fused other cell with this cell.
3. 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
1. source of DNA with gene of interest
2. vector (our prokaryotic/low form of DNA)
3. sev bacterial enzymes & processes
4. 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
1. extract sample DNA (complementary DNA; comp to mRNA), genomic, chromosome specific)
2. cut sample DNA and vector DNA with same restriction enzyme: plasmid, phage, cosmid or YAC
3. Mix, and ligate
4. Transform/transfect into host: transformation/transduction
5. plate on selective media
6. make replicate
7. screen for specific DNA fragment.
what are the sources of human DNA containing the gene you wish to clone?
1. whole genomic DNA
2. chromosome specific DNA
3. 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:
1. plasmids: <5-10kb
2. bacteriophage: 20kb
3. cosmids: 50 kb
4. yeast artificial chromosomes; linear, eukaryotic; 100-1000kb
what are features of pBR322 plasmid?
1. R genes
3. 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:
1. Lytic: makes lots of copies of themselves and make new protein capsids and lyse cell and kill host.
2. 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
3. RE sites: EcoRI, BamHI,
4. antibiotic selectable markers
what are features of Type II restriction endonucleases?
1. bacterial defense mechanism
2. DS DNA seq specificity; rec 2-8 bp palindromes (read same way backwards and forwards)
3. many produce sticky ends
4. 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?
1. 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
1. coned DNA fragment from another species
2. Anitbody specific for gene product
3. PCR amplified DNA fragment
4. 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?
1. Seq it: DNA seq det. protein seq, so many clues about protein function can be derived from the DNA seq of its gene.
2. Express it: Recomb proteins can by synthesized (how we get insulin, human GH)
3. Use it as a probe: use to detect mutations in genes. Use cloned minisatellite seq as probes to prod DNA fingerprints for ind identification
4. Use it in functional studies: detect mRNA exp of the gene in tissues, ind cells, in response to sig molecules
5. transfer it: gene therapy correct a genetic defect.
What are applications of cloned DNA sequences
1. sequence analysis
2. molecular probes: southern blots: forensics/patient diagnosis
northern blots: gene exp studies
in situ hybridization
3. 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.
1. 4 rx mixes are set up; each has a diff ddNTP analog + 35S labeled dNTPs
2. ddNTP analog is added in a specific low ddNTP: dNTP ratio
3. since dNTPs >> ddNTPs, synthesis will terminate at various points in seq.
4. 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?
1. 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)
2. fusion protein production: can follow protein around
3. in vitro translation: to protein product
4. 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.