Chapter 19: Molecular Genetic Analysis and Biotechnology Flashcards
restriction enzymes
enzymes that recognize specific nucleotide sequences in DNA and make double-stranded cuts at those sequences
- produced naturally by bacteria and used to fight viruses
- palindromic sequences
- bacteria protects its own DNA by methylation
cohesive/sticky ends
short, single stranded overhanging end on a DNA molecule produced when DNA is cut by certain restriction enzymes
- complementary
- can spontaneously pair to rejoin DNA fragments that have been cut with the same restriction enzyme
CRISPR-Cas System
molecular tool for precisely cutting DNA
- occurs naturally in bacteria and archaea
- when foreign DNA enters the cell, proteins cut up the foreign DNA and insert bits of it into a CRISPR array, which then serves as a memory of the invader
- array is transcribed and cleaved into crRNA which combines with proteins to form effector complexes
advantages and limitations of CRISPR
advantages:
- genome editing
- precise and unique cuts
- potential for genetic engineering and biotechnology
limitations:
- potential for off target cleavage
- potential to create genetic mosaics
- getting the components into a cell
gel electrophoresis
electrophoresis used to separate DNA molecules
- separates molecules based on size and charge
- wells are made in agarose gel, solutions of DNA fragments are placed in the wells and electrical current is passed through the gel
- DNA fragments migrate towards the positive end of the gel
- small DNA travels quicker than large, so the fragments separate based on size
- stains
probe
DNA or RNA molecule with a base sequence complementary to a sequence in the gene of interest
- bases will pair with the bases on a complementary sequence and locate a specific gene or another DNA sequence
southern blotting
technique by which DNA is transferred from gel to a solid support, such as a nitrocellulose or nylon filter
northern blotting
process by which RNA is transferred from a gel to a solid support, such as a nitrocellulose or nylon membrane
western blotting
transfer of protein to a solid support
gene cloning
identical copies of the original piece of DNA are replicated within bacterial cells
polymerase chain reaction (PCR)
DNA replication catalyzed by a DNA polymerase
- amount of DNA doubles with each replication event
- allows fragments to be amplified a billionfold within a few hours
- can be used with very small amounts of DNA
cloning vector
a stable, replicating DNA molecule to which a foreign DNA fragment can be attached for introduction into a cell
- has three important characteristics:
1. origin of replication
2. selectable marker
3. one or more unique restriction sites into which a DNA fragment can be inserted
plasmids
circular DNA molecules that exist naturally in bacteria
- commonly used as vectors for cloning DNA fragments in bacteria
- able to replicate independently of the bacterial chromosome
transformation
process in which DNA (plasmid) is taken up by a bacterial cell
cosmids
plasmids that are packaged into empty viral protein coats and transferred to bacteria by viral infection
bacterial artificial chromosomes (BACs)
vectors originally constructed from the F plasmid and can hold very large fragments of DNA
expression vector
cloning vector containing DNA sequences such as a promoter, a ribosome-binding site, and transcription initiation and termination sites that allow DNA fragments inserted into the vector to be transcribed and translated
DNA library
a collection of clones containing all the DNA fragments from one source
- can be screened to find a gene or sequence of interest
genomic library
the set of bacterial colonies or phages containing DNA fragments from human cells
cDNA library
a library consisting only of those DNA sequences that are transcribed into mRNA
- enriched with fragments from actively transcribed genes
- introns do not interrupt cloned sequences
in situ hybridization
method used to determine the chromosomal location of a gene or another specific DNA fragment or the tissue distribution of an mRNA by using a labeled probe that is complementary to the sequence of interest
- cell must be fixed and the chromosomes be spread on a microscope slide and denatured
- probes are added that fluoress to investigate different sequences
positional cloning
isolation of genes on the basis of their position on a gene map
restriction fragment length polymorphisms (RFLPs)
genetic markers useful for mapping
DNA sequencing
determines the sequence of bases in a DNA molecule
dideoxy sequencing
special nucleotides called ddNTPs are used as substrates
- after a ddNTP has been incorporated into the strand, no more nucleotides can be added because there is no 3’-OH group to form a phosphodiester bond
DNA fingerprinting
use of DNA sequences to identify individual people
microsatellites or short tandem repeats (STRs)
very short DNA sequences repeated in tandem
- found at many loci throughout the human genome
forward genetics
approach that begins with a phenotype and proceeds to a gene that encodes the phenotype
reverse genetics
approach that begins with a genotype and proceed to the phenotype by altering the sequence or inhibiting its expression
targeted mutagenesis
process where mutations are induced at specific locations
site-directed mutations
strategy for mutation where you cut a short sequence of nucleotides with restriction enzymes and replace it with a synthetic oligonucleotide
oligonucleotide-directed mutagenesis
mutation method where a single-stranded oligonucleotide is produced that differs from the target sequence by a few bases.
- target nucleotide and oligonucleotide will pair
- olgonucleotide acts as a primer to initiate DNA synthesis
RNAi
RNA that has potential to be a therapeutic agent for the treatment of human diseases
- can temporarily turn off genes and observe the phenotype
- Fire and Mellow (1998)
