L-3 and L-4 Amplification of DNA and RNA sequences, and Sanger sequenceing Flashcards
what are the advantages of polymerase chain reaction
sensitive, fast, and safe (non radioactive), multiple copies, and has several applications
what are the disadvantages of polymerase chain reaction
contamination, finicky for long DNA segments, may introduce mutations
what are the three main steps involved in PCR
Denaturation at 95C, annealing of primers (50-70C), synthesis of new DNA at 70C
what are the components of PCR
dsDNA template, two (ss oligos) primers, thermostable DNA polymerases, dNTPs, Mg2+ and buffer
things to remember when designing primers
sequences should always correspond to the 5’ end of sequences of each strand of template DNA and always written 5’ to 3’
What factors may impact the Tm
GC content, secondary structure, primer dimers, and primers should have a Tm of +/- 5C
Considerations when adding sequences in template DNA
seq always added to 5’ end of primers, added seq will not anneal in first cycle, after multiple rounds the majority will have additional sequences
What does site directed mutagenesis involve
used to alter specific sequences; PCR reactions in which primers are designed to have complementary sequences flanking the regions but have different bases at mutation sites.
How are non-mutants removed following PCR
digestion with DpnI (specific for methylated DNA); synthetic DNA does not posses methylation and is not targeted/detected
how is RNA amplified?
cannot be directly cloned; a cDNA molecule must be made with reverse transcriptase followed by conventional PCR; RQs a single primer
Important caveat with RNA amplification
cannot gain information about non-coding regions
what are required components for DNA vectors
sequences that allow replication in host, Selectable markers, and (optional) multiple cloning sites
What sequences are recognized by REs
specific short palindromic DNA sequences
which bond is cleaved by REs
the 3’OH
why is the restriction modification system important
REs are paired with methylases in vivo so that the host organism does not cut up its own DNA
Describe the process of cloning by ligation
RE generate compatible ends, phosphodiester bonds are forms by ligases, (competent) bacteria is transformed, cells are cultured and selected; RQ atp
Describe the process of cloning by recombination
sites are added the same as RE sites; occurs between two homologous regions of DNA molecules, different sequences at two ends of insert control direction
Advantages and disadvantages of cloning by recombination
only recombinase is needed for the reaction however the sequences must be able to be recognized by recombinase
Describe Gibson Cloning
exonucleases are used in creating overhangs and fragments are designed to have 20-40 bp end sequences that match the sequences of other fragments and vector; can be use to assemble large genomes
the three steps involved in gibson cloning
exonucleases create the 5’ or 3’ overhang, dna polymerase fills in gaps, dna ligase joins the fragments
what is a genomic library
a library that contains clones of all DNA
what is a cDNA library
a set of clones that represent mRNAs in a given cell type
Advantages of using a cDNA library
the libraries are different based on a given tissue type; it is possible to identify and study genes in a biological context; one can compare the same genes depending on the tissue type
Describe sanger sequencing (chain terminated dideoxy sequencing)
a single primer anneals to a sequence of interest and the primer is extended with DNA polymerase 1, after extension the reactions stops once a dideoxy nucleotide is incorporated (no 3’OH). The end result is a matrix of molecules of different sizes
