DNA Analytical techniques Flashcards
1
Q
restriction enzyme
A
- Endonuclease enzymes that recognize specific base sequences in double-stranded DNA
- Cleave both strands of the duplex at specific places.
2
Q
DNA probe
A
- A radioactively labeled, single-stranded specific base sequence
- Used to locate a complementary sequence among DNA fragments displayed on an electrophoretic gel.
3
Q
Southern blotting
A
- A technique used to locate and identify a DNA fragment containing a specific sequence;
- a mixture of fragments is separated by electrophoresis,
- transferred to a nitrocellulose sheet,
- hybridized to a radioactively labeled DNA probe complementary to the desired sequence,
- Visualized by autoradiography.
4
Q
northern blotting
A
- A technique analogous to Southern blotting,
- A mixture of RNA fragments is separated by electrophoresis,
- transferred to a nitrocellulose sheet,
- hybridized to a radioactively labeled DNA probe complementary to the desired sequence,
- visualized by autoradiography.
- The technique can therefore be used to locate and identify an RNA fragment containing a specific sequence.
5
Q
controlled termination of replication (Sanger dideoxy method)
A
- A DNA-sequencing technique that employs controlled interruption of enzymatic replication of the molecule to be analyzed.
- DNA polymerase I is used with a primer,
- the four deoxynucleoside triphosphates, and
- a 2’, 3’-dideoxy analog of one of them.
- Fragments of various lengths are produced in which the dideoxy analog is at the 3’ end.
- Four sets of chain-terminated fragments (one for each analog) are then displayed by electrophoresis and autoradiography
- the base sequence can be read from the four lanes of the gel.
6
Q
polymerase chain reaction (PCR)
A
- A method for amplifying DNA sequences using DNA polymerase;
- a series of three-step cycles is employed,
- in which parental DNA strands are separated by heating,
- primers to flanking regions of the target sequence are annealed to the separated strands,
- the primers are then extended by DNA synthesis.
7
Q
vector
A
- A DNA molecule that can replicate autonomously in an appropriate host organism.
- Vectors are designed to enable the rapid, covalent insertion of DNA fragments of interest.
8
Q
plasmid
A
- Circular duplex DNA molecules that replicate autonomously
- act as accessory chromosomes in bacteria;
- they carry useful genes but are disposable under certain conditions.
9
Q
cloning vector
A
- These are plasmids or bacteriophage that allow the insertion and replication of DNA fragments into bacteria for the purpose of cloning.
- They often feature a polylinker region that includes many unique restriction sites within its sequence,
- allowing the region to be cleaved with many different restriction enzymes or combinations of enzymes.
- This provides great versatility in the DNA fragments that can be inserted.
10
Q
reporter gene
A
- Genes which encode readily-detectable markers such as:
- antibiotic-resistance enzymes
- fluorescent proteins.
11
Q
expression vector
A
- A plasmid-cloning vector
- Has been optimized for the expression of large amounts of recombinant protein encoded by the vector.
12
Q
lambda (λ) phage
A
- A bacteriophage cloning vector that can be incorporated into the hosts genome
- Thus can be replicated indefinitely
- Or can be expressed and destroy the host.
13
Q
bacterial artificial chromosome (BAC)
A
- An artificial bacterial chromosome,
- a highly engineered version of the E. coli fertility (F factor), that can serve as a cloning vector for inserts as larges as 300 kb.
14
Q
yeast artificial chromosome (YAC)
A
- A DNA molecule that can be used to clone DNA inserts ranging from 100 to 1000 kb in length;
- these molecules contain a centromere,
- an autonomously replicating sequence,
- a pair of telomeres,
- selectable marker genes,
- an insertion site for the sequence to be cloned.
15
Q
genomic library
A
- A collection of DNA fragments,
- inserted into vector molecules,
- Represents the entire genome of an organism.
16
Q
complementary DNA (cDNA)
A
- DNA complementary to an mRNA sequence.
18
Q
site-directed mutagenesis
A
- A method in which a primer containing a mismatched nucleotide is used to produce a desired change in a DNA sequence.
- It can readily produce mutant proteins with single amino acid substitutions.
19
Q
cassette mutagenesis
A
- A means of introducing a variety of mutations into a gene of interest.
- A short segment of plasmid harboring the original gene is removed by restriction enzyme treatment.
- A synthetic double-stranded oligonucleotide (the cassette) carrying the genetic alterations of interest is subsequently inserted.
20
Q
next-generation sequencing
A
- Platforms that enable the rapid determination of a complete genome sequence
- Combining breakthroughs in the handling of very small amounts of liquid,
- high-resolution optics,
- computing power.
21
Q
quantitative PCR (qPCR)
A
- A polymerase chain reaction-based technique
- For determining the amount of individual mRNA molecules present in a population of RNA molecules.
22
Q
DNA microarray (gene chip)
A
- A solid support such as a microscope slide to which are affixed oligonucleotides or cDNAs corresponding to specific genes.
- Fluorescently labeled cDNA is hybridized to the slide
- Reveals the expression level for each gene,
- Identifiable by its known position within the microarray.
25
Q
genome editing
A
- Highly specific modification of genomic DNA.
26
Q
RNA interference
A
- The suppression of the transcription of a gene
- following the introduction into the cell of double stranded RNA molecule that contained sequences present in the suppressed gene.
28
Q
tumor-inducing plasmid (Ti plasmid)
A
- Plasmids carried by A. tumefaciens
- Encode instructions for induction of tumor state in infected plants cells.
- The resulting tumor tissue is called a crown gall.
