L-3 and L-4 Amplification of DNA and RNA sequences, and Sanger sequenceing

Question 1

what are the advantages of polymerase chain reaction

Answer 1

sensitive, fast, and safe (non radioactive), multiple copies, and has several applications

Question 2

what are the disadvantages of polymerase chain reaction

Answer 2

contamination, finicky for long DNA segments, may introduce mutations

Question 3

what are the three main steps involved in PCR

Answer 3

Denaturation at 95C, annealing of primers (50-70C), synthesis of new DNA at 70C

Question 4

what are the components of PCR

Answer 4

dsDNA template, two (ss oligos) primers, thermostable DNA polymerases, dNTPs, Mg2+ and buffer

Question 5

things to remember when designing primers

Answer 5

sequences should always correspond to the 5’ end of sequences of each strand of template DNA and always written 5’ to 3’

Question 6

What factors may impact the Tm

Answer 6

GC content, secondary structure, primer dimers, and primers should have a Tm of +/- 5C

Question 7

Considerations when adding sequences in template DNA

Answer 7

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

Question 8

What does site directed mutagenesis involve

Answer 8

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.

Question 9

How are non-mutants removed following PCR

Answer 9

digestion with DpnI (specific for methylated DNA); synthetic DNA does not posses methylation and is not targeted/detected

Question 10

how is RNA amplified?

Answer 10

cannot be directly cloned; a cDNA molecule must be made with reverse transcriptase followed by conventional PCR; RQs a single primer

Question 11

Important caveat with RNA amplification

Answer 11

cannot gain information about non-coding regions

Question 12

what are required components for DNA vectors

Answer 12

sequences that allow replication in host, Selectable markers, and (optional) multiple cloning sites

Question 13

What sequences are recognized by REs

Answer 13

specific short palindromic DNA sequences

Question 14

which bond is cleaved by REs

Answer 14

the 3’OH

Question 15

why is the restriction modification system important

Answer 15

REs are paired with methylases in vivo so that the host organism does not cut up its own DNA

Question 16

Describe the process of cloning by ligation

Answer 16

RE generate compatible ends, phosphodiester bonds are forms by ligases, (competent) bacteria is transformed, cells are cultured and selected; RQ atp

Question 17

Describe the process of cloning by recombination

Answer 17

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

Question 18

Advantages and disadvantages of cloning by recombination

Answer 18

only recombinase is needed for the reaction however the sequences must be able to be recognized by recombinase

Question 19

Describe Gibson Cloning

Answer 19

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

Question 20

the three steps involved in gibson cloning

Answer 20

exonucleases create the 5’ or 3’ overhang, dna polymerase fills in gaps, dna ligase joins the fragments

Question 21

what is a genomic library

Answer 21

a library that contains clones of all DNA

Question 22

what is a cDNA library

Answer 22

a set of clones that represent mRNAs in a given cell type

Question 23

Advantages of using a cDNA library

Answer 23

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

Question 24

Describe sanger sequencing (chain terminated dideoxy sequencing)

Answer 24

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

Question 25

How is the matrix of DNA analyzed

Answer 25

by size and with the radioactively labeled dDNA; the polynucleotides pass through capillary gel electrophoresis

Question 26

advantages and disadvantages of sanger sequencing

Answer 26

RQs prior knowledge about the sequence (to produce a primer) and the resolution is impacted by size however this is the most accurate method of sequencing