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Collections of cloned DNA fragments from a particular organism contained in plasmid vectors within host bacteria (ex. E. colt
Genomic Libraries: What happens to Chromosomal DNA?
Genomic Libraries: What happens to the vector (plasmid)?
Genomic Libraries: What happens to DNA ligase?
Genomic Libraries: What happens to recombinant vectors?
used to transform bacteria, and theoretically each bacteria will contain a single recombinant plasmid
cDNA libraries need to make double stranded DNA from mRNA: How?
Short linker double stranded DNA sequences, which contain restriction enzyme recognition sites, are added to the ends of the cDNA.
Cut with restriction enzyme, cut vector with same enzyme, ligate fragments to create recombinant vectors
Then transform bacteria with recombinant vectors
colony hybridization 1st step
colony hybridization 2nd step
colony hybridization 3rd step
Treat filter with alkaline solution to lyse the cells and denature the DNA; then bake filter or UV exposure
colony hybridization 4th step
colony hybridization 5th step
Filter is incubated with a probe that is tagged with a radioactive nucleotide or fluorescent dye
colony hybridization 6th step
Probe binds by hydrogen bonding to complementary sequences on the filter = hybridization
colony hybridization 7th step
colony hybridization 8th step
Filter exposed to X-rav film or digital camera to detect fluorescent probe
fluorescent probe 9th step
Film or picture is then compared to the original agar
plate to identify which colonies contained recombinant
plasmid with the gene of interest
no. Usually get small pieces of the gene: the pieces are sequenced and scientists look for overlapping sequences.
Technique for making copies, or amplifying, a specific sequence of DNA in a short period of time
1983 by Kary Mullis polymerase chain reaction (PCR)
PCR 1st step
Target DNA to be amplified is added to a tube,mixed with nucleotides (dATP, dCTP, dGTP, dTTP)., buffer with MgCl2, and DNA polymerase.
PCR 2nd step
Paired set of Forward and Reverse Primers are added - short single-stranded DNA oligonucleotides (18-22 nucleotides long)
PCR 3rd step
Reaction tube is placed in an instrument called a thermocycler
through a series of reactions called a PCR cycle
(hybridization) - in which primers H bond with complementary bases at the opposite ends of target sequence at 52 °C to 58 °C
(elongation) - DNA Pol copies target
DNA at 70 to 75 °C
amplify millions of copies of target DNA from small amount of starting material in short period of time
To calculate the number of copies of target DNA starting with 1 molecule of DNA use this equation 2N in which N represents number of PCR cycles
Assume you want to do 2 PCR cycles to amplify your DNA insert, how many copies of DNA will you have at the end of your PCR?
2 to the power of 2. 4.
isolated from a species known as Thermus aquaticus that thrives in hot springs
- Making DNA probes
- Studying gene expression
- Detection of viral and bacterial infections
-Diagnosis of genetic conditions
-Detection of trace amounts of DNA from tissue found at crime scene
- Detection of DNA from fossilized tissue
Need to know something about the DNA sequence that flanks the gene of interest to design primers
polymerase puts a single adenine nucleotide on the 3' end of all PCR products
Sanger method. Important to determine the sequence of nucleotides of the cloned gene
-single primer annealing to denatured DNA template
- all 4 dNTPs
-DNA Polymerase
-dideoxynucleotide (ddNTP) which has a 3' H instead of 3'OH on the deoxyribose so it cannot form a phosphodiester bond with the incoming nucleotide & so gets terminated
Original Sanger Method 1st step
Had four separate reaction tubes and each contained the same DNA; radioactively labeled single primer; all 4 dNTPs; and a small amount of one daNTP per tube; DNA Pol
Old Sanger Method 2nd step
Over time a ddNTP will be incorporated into all the positions in the newly synthesized strands creating fragments of varying lengths that are terminated at the ddNTP
Original Sanger Method 3rd step
Original Sanger Method 4th step
complimentary to the sequence on the template strand in the vector
enables greater than 600 nucleotides to be sequenced per reaction. very helpful for completing human genome project.