Lecture 11 Flashcards
Steps of Molecular Cloning
1A) Digest: cut vector DNA and DNA to be cloned w/ same restriction enzyme (s). 1B) Ligation: mix DNA and ligate w/ DNA ligase to create recombinant DNA molecule. 2A) Transformation: transfer recombinant DNA into host cells. 2B) Selection and Growth: Plate and grow transformed calls on selective media.
DNA Library
Long-lived collection of cellular clones containing many different DNA fragments. Each clone carries a different DNA fragment inserted into a vector
Genomic DNA library
Collection of clones that contain inserted DNA fragments that, collectively, include all the genomic DNA of an organism.
cDNA
Complimentary DNA, a fragment of DNA that is complementary to an mRNA
Mechanism of Reverse Transcription
1) Oligo dT primer anneals to poly A tail of mRNA. 2) Reverse transcriptase reads mRNA, creates a DNA strand complementary to mRNA. 3) Creates a double-stranded hybrid RNA/DNA
Mechanism of second strand synthesis
RNA is degraded with RNase. 3’ end of DNA folds back on itself, acts as a primer; DNA polymerase is added and creates DNA strand complementary to first strand. S1 nuclease is added; it cuts hairpin loop. Results in a double stranded DNA with a cDNA strand attached to a strand with the same sequence as mRNA except has T’s instead of U’s.
Steps of constructing a cDNA library.
1) mRNA is isolated from cells, usually from a particular tissue or particular developmental stage. 2) Reverse transcription: DNA copy of each mRNA is made 3) Second-strand DNA synthesis: Creates a double stranded cDNA for each original mRNA, one strand complementary to original mRNA, one strand same sequence as original mRNA. 4) cDNA are utilized in molecular cloning. Creates a set of clones that collectively contain DNA copies of all the mRNAs found in the original tissue.
DNA Primers
short (18-24 nucleotides) single-stranded DNAs. Complementary to 3’ end of strand to be copied in PCR.
3 Steps in each cycle of PCR
Dnaturation, annealing, extension
Denaturation (PCR)
Heat breaks H-bonds and separates DNA strands.
Annealing (PCR)
Base pairing of primers to 3’ ends of DNA strands to be copied
Extension (PCR)
Creation of complementary DNA strands starting at 3’ end of primers by DNA polymerase
Required Components of PCR
DNA template containing sequence to be amplified. Could be genomic, plasmid or cDNA. Two DNA primers, specific to ends of DNA fragment to be amplified. Each corresponds to one of the two DNA strands. PCR Buffer. Mg2+ helps determine specificity of annealing of primers to template. Taq polymerase. thermostable DNA polymerase. dNTPs (all four deoxynucleotides). Nuclease-free H2O
Typical PCR Reaction Conditions
Initial denaturation step: 95C for 2 min. PCR cycles through steps: Denature at 95C for 30 sec, anneal primers 45-65C for 30 sec, extension: Dna polymerase extends DNA from primer, reading DNA template 72C for 30 sec. Final Extension, ensures all new DNA fragments are full length 72C for 10 min.
