Lecture 16 - Methods of nucleic acid analysis Flashcards
DNA sequencing determines
the exact sequence of a DNA molecule
In solid-phase synthesis of nucleic acids,
sequences of nucleic acids can be synthesized and used to identify or amplify other nucleic acids
Restriction enzymes
“cut” precise DNA segments allowing for manipulation of a given sequence
Blotting techniques are used to
separate and characterize DNA (southern) and RNA (western)
Fluorescent in situ hybridization (FISH)
locates DNA/RNA sequences in the cell
The polymerase chain reaction
amplifies a segment of DNA
Microarrays
quantify the expression of genes
RNAseq
quantifies the copy number of each RNA molecule in a sample
Sanger sequencing is based on
generation of DNA fragments whose length depends on the last base in the sequence
In Sanger sequencing, collections of DNA fragments can be generated by
controlled termination of replication (Sanger dideoxy method)
The four reaction mixtures in Sanger sequencing all contain
- DNA polymerase
- DNA primer
- Radiolabeled dATP, TTP, dGTP, dCTP
- 2’, 3’-dideoxy analog
In Sanger sequencing, a chemically synthesized primer is required for the DNA polymerase to
start synthesizing the complementary strand
In Sanger sequencing, most of the new growing DNA molecules consist of
one of the four deoxyribonucleosides (they’re in excess)
In Sanger sequencing, occasionally, a
dideoxy analog will be incorporated into the growing chain (blocks further growth)
Why isn’t Sanger sequencing used for sequencing the entire genome?
Not fast enough
Developing automatic DNA sequencers based on ___________ allowed more rapid sequencing of larger amounts of DNA
fluorescent dideoxynucleotide chain terminators
Pseudogenes
resemble functional genes but are no longer expressed
Pseudogenes are formerly functional genes that
picked up mutations
Pseudogenes are transcribed into
RNA and function to regulate parental genes
Solid-phase DNA synthesis can
immobilize the growing product while flushing soluble contaminants and by-products
Steps in solid-phase DNA synthesis
- 3’phosphorus atom of incoming nucleotide is joined to 5’ oxygen fo growing chain
- Phosphate triester is oxidized by iodine
- Removal of DMT protecting group on 5’-OH of growing chain
- NH3 is added to remove remaining protecting groups
Restriction enzymes recognize
specific sequences in dsDNA and cleave the DNA at a specific place
Restriction enzymes can be used to
- Analyze chromosome structure
- Sequence long DNA molecules
- Isolate genes
- Create new DNA molecules
Restriction enzymes cleave by
hydrolyzing a phosphodiester bond in each strand of DNA
Cleavage sites in restriction enzymes are often
palindromic
Restriction enzymes selectively degrade
foreign DNA but not their own DNA
Restriction enzymes may cut DNA resulting in
- sticky ends
- blunt ends
A specific gene or fragment of DNA may be cleaved several times, creating a
pattern of restriction fragments that are unique
Gel electrophoresis separates molecules based on
size and charge
What charge does DNA have?
negative
In gel electrophoresis, DNA is usually stained with
ethidium bromide
Radioactive DNA can be detected by
autoradiography
Polyacrylamide gels are used to
separate fragments containing up to 1000 bp
Agarose gels are used to
resolve mixtures of larger fragments (up to 20 kb)
Which is more porous, polyacrylamide gel or agarose gel?
Agarose gel
A Southern blot is based on
hybridization of the DNA fragment of interest with a labeled complementary DNA strand
Type of blots used with Southern blotting
DNA
Type of blots used with Northern blotting
RNA
Type of blots used with Western blotting
Protein
Fluorescent in situ hybridization (FISH) is a
cytogenetic technique
FISH uses
fluorescent probes that bind to the parts of a chromosome with a high sequence complementarity
FISH can be used in identifying specific features in
DNA
3 steps of PCR
- Strand separation
- Hybridization of primers
- DNA synthesis
In PCR, the two strands of DNA are denatured by
heating
In PCR, the primers are hybridized by
cooling the solution
In PCR, the solution is heated to _______ for DNA synthesis
72 degrees Celsius
What is mixed in PCR?
- DNA to be amplified (template)
- DNA polymerase (Taq)
- primers
- dNTPs
A great advantage of PCR is the
exponential amplification of DNA molecules
Quantitative PCR (qPCR) is used to investigate
mRNA levels
The fluorescent reporter molecule used in qPCR reactions is typically
SYBR Green
In probe-based qPCR, a fluorescent reporter dye is attached to
the 5’ end of a probe
In probe-based qPCR, a quencher is attached to the
3’ end of a probe
RNA-seq (RNA sequencing) uses next-generation sequencing (NGS) to
reveal the presence and quantity of RNA in a biological sample at a given moment in time
