PCR, Mutagenesis, DNA and RNA-Seq Flashcards
What is the function of DNA hybridization?
identifies a specific DNA sequence within a DNA sample
How are DNA fragments cut? (Southern Blot Analysis)
cut with specific restriction endonucleases
How are different fragments separated? (Southern Blot Analysis)
separated by size with gel electrophoresis
How are DNA fragments denatured? (Southern Blot Analysis)
with an alkali to expose the bases and are transferred to a membrane
What happens to the DNA fragment after it is transferred to a membrane? (Southern Blot Analysis)
membrane is probed with a radioactively or otherwise labeled single stranded DNA molecule that is complementary to the target DNA
What is the function of the probe? (Southern Blot Analysis)
hybridizes with any DNA fragments containing the target sequence. These fragments can be identified by autoradiography, chemiluminescence, or fluorescence
What is the purpose of Northern Blot Analysis?
gene expression is most often controlled at the level of transcription so it is useful to examine RNA levels
How are RNAs separated? (Northern Blot Analysis)
separated by denaturing gel electrophoresis, transferred to a membrane for probing with a leveled DNA (or RNA) probe that is complementary to the RNA (gene) of interest
What is the function of hybridization of the probe to the RNA? (Northern Blot Analysis)
identifies transcript and reveals its size
What are the base requirements for DNA synthesis? (4)
- a single-stranded DNA template
- an annealed (complementary) primer with a 3’ OH
- all four dNTPs (dATP, dCTP, dGTP, dTTP)
- DNA polymerase
What is the function of DNA Polymerase?
catalyzes DNA synthesis by activating 3’ OH
What is nucleophilic displacement?
involves the movement of an electron from an electron-rich atom (nucleophile) to an electron-poor atom (electrophile)
When is nucleophile attacked encouraged?
when a proton is removed by a general base on an enzyme – here the H+ on the 3’ OH is removed
What do enzymes like DNA Polymerase provide?
groups that act as acids or bases and encourage proton movements – acid-base catalysis
What is the function of PCR?
amplification of large amounts of a specific or random DNA from a small sample for various purposes
What are the purposes of PCR? (7)
- cloning
- sequencing
- making probes
- gene mapping
- gene expression analysis
- mutagenesis
- diagnostics/forensics
What is the amplification from n cycles?
2^n, assumption: 100% of all templates in every cycle
What are the molecular requirements for PCR?
- template DNA
- primer pair complementary to DNA sequences that flank the region to be amplified, correctly oriented
- DNA Polymerase (thermostable)
- dNTPs
What are the 3 steps of PCR cycle?
- denaturation (of templates) ~95 C
- annealing (of primers to template) ~50 C
- extension (of primers by thermostable polymerase) ~72 C
How often does PCR repeat?
many times (20-40 times)
T or F: primers can modify PCR’d sequences
true
What is PCR used for? (2)
- use to faithfully amplify a specific piece of DNA
- introduce mutations to to add sequences to the ends of molecules
What are the cycles of in PCR?
- added sequences will not anneal in the first PCR cycle, but will be present in the template for subsequent cycles
- after multiple rounds of PCR< the vast majority of molecules have the additional sequences at the 5’ and 3’ ends
What is Site-Directed Mutagenesis used for?
to alter the DNA sequence at specific locations. This procedure can be useful in for creating specific mutant proteins to learn more about their functions
How is Site-Directed Mutagenesis performed?
using PCR reactions in which the primers are designed so they have a complementary sequence to the flanking regions but have different bases at the mutation site, shown as a V in the image
What follows PCR reactions (Site-Directed Mutagenesis)?
non-mutants are removed by digestion and the desired mutant molecules used for transformation
What are the steps of site-directed mutagenesis-PCR?
- two short PCR products are made, each with a mutation near the middle gene
- two short PCR products are mixed generating a full length mutated gene
- another PCR reaction is then performed containing two primers that anneal to the ends of the gene
- two primers used to introduce the mutation are complementary so the short PCR products act as primers in the final PCR product
What is an epitope?
the sequence of amino acids recognized by any antibody
What is the function of Tag sequences?
may be added to the N or C termini of proteins to enable their analysis
What is the structure of dideoxynucleotides?
lack the 2’OH and 3’OH
What is the function of dideoxynucleotides?
blocks further polymerizaiton because there will be no 3’OH
What are the steps to the chain termination by dideoxynucleotides?
- in the lower panel, dideoxycytidine (ddC) was incorporated instead of dC
- the absence of a the 3’OH means there is no reactive OH to join to the next nucleotide
- chain stops growing and the chain length can be measured
What are the steps of DNA sequencing by Chain Termination?
- a primer is annealed to a DNA molecule of interest
- in the presence of all four normal dNTPs and DNA polymerase, the primer will be extended to the end of the template
- in the additional presence of a tiny bit of a dideoxynucleotide, chains will stop growing when a dideoxynucleotide is incorporated, indicating the position of its complementary base
How many reactions are performed in DNA sequencing by chain termination?
four different reactions, each with a different dideoxynucleotide: ddA, ddC, ddG, and ddT
What is the function of denaturing polyacrylamide gel?
allows for dideoxynucleotide reactions to run, which allows analysis of the single synthesized strand with single base resolution
What does the position of a band indicate (in polyacrylamide gel)?
indicates where each base was incorporated
How can DNA sequence be determined using chain-terminated dideoxy sequencing?
- a single primer (turquoise) is annealed to the sequence of interest and the primer extended with DNA polymerase I
- the reaction mixture contains all four deoxy bases (dATP, dTTP, dCTP, dGTP), each with a different color
- the extension of polynucleotide will stop if dideoxy nucleotide is included in the chain as no 3’ OH group will be available on the sugar
- generates a set of molecules of different sizes each terminating at a dideoxynucleotide
How are labeled polynucleotides separated?
by size with capillary gel electrophoresis and measure the color of each size fragment with a laser (displayed on a chromatogram)
How can mRNA be converted into complementary DNA?
- Oligo-dT is used to prime DNA polymerization by reverse transcriptase using mRNA as template and producing a complementary strand
- DNA strand is copied by DNA polymerase using random primes to make double-stranded cDNA, which lacks most of the non-coding regions of the gene (introns are gone)
What is the difference between mRNA and RNA?
- mRNA is <5% of total RNA but is the most “interesting” for gene expression
- normally poly-adenylated at the 3’ end; this feature is used to isolate mRNA from total RNA
What can PCR quantify?
the amount of cDNA as the amount of template for the PCR reaction will determine the amount of product as long as the PCR reaction is in linear range of amplificaiton
Primers are chose to amplify the _ of an mRNA
cDNA copy
What is the difference between mRNA and qPCR?
allows for determination of mRNA amount and is more sensitive than Northern blot but still analyzes one gene at a time
What is transcriptome?
the collection of all transcripts in the cell
What is mRNA sample converted into?
cDNA
What is cDNA converted into?
a library for sequencing by Illumina or other high-throughput sequencing method
How are each mRNAs represented in RNA sequencing?
the number of times it is separately sequenced in the analysis
