Chapter 19: Molecular Genetic Analysis and Biotechnology

Question 1

restriction enzymes

Answer 1

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

Question 2

cohesive/sticky ends

Answer 2

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

Question 3

CRISPR-Cas System

Answer 3

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

Question 4

advantages and limitations of CRISPR

Answer 4

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

Question 5

gel electrophoresis

Answer 5

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

Question 6

probe

Answer 6

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

Question 7

southern blotting

Answer 7

technique by which DNA is transferred from gel to a solid support, such as a nitrocellulose or nylon filter

Question 8

northern blotting

Answer 8

process by which RNA is transferred from a gel to a solid support, such as a nitrocellulose or nylon membrane

Question 9

western blotting

Answer 9

transfer of protein to a solid support

Question 10

gene cloning

Answer 10

identical copies of the original piece of DNA are replicated within bacterial cells

Question 11

polymerase chain reaction (PCR)

Answer 11

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

Question 12

cloning vector

Answer 12

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

Question 13

plasmids

Answer 13

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

Question 14

transformation

Answer 14

process in which DNA (plasmid) is taken up by a bacterial cell

Question 15

cosmids

Answer 15

plasmids that are packaged into empty viral protein coats and transferred to bacteria by viral infection

Question 16

bacterial artificial chromosomes (BACs)

Answer 16

vectors originally constructed from the F plasmid and can hold very large fragments of DNA

Question 17

expression vector

Answer 17

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

Question 18

DNA library

Answer 18

a collection of clones containing all the DNA fragments from one source
- can be screened to find a gene or sequence of interest

Question 19

genomic library

Answer 19

the set of bacterial colonies or phages containing DNA fragments from human cells

Question 20

cDNA library

Answer 20

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

Question 21

in situ hybridization

Answer 21

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

Question 22

positional cloning

Answer 22

isolation of genes on the basis of their position on a gene map

Question 23

restriction fragment length polymorphisms (RFLPs)

Answer 23

genetic markers useful for mapping

Question 24

DNA sequencing

Answer 24

determines the sequence of bases in a DNA molecule

Question 25

dideoxy sequencing

Answer 25

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

Question 26

DNA fingerprinting

Answer 26

use of DNA sequences to identify individual people

Question 27

microsatellites or short tandem repeats (STRs)

Answer 27

very short DNA sequences repeated in tandem
- found at many loci throughout the human genome

Question 28

forward genetics

Answer 28

approach that begins with a phenotype and proceeds to a gene that encodes the phenotype

Question 29

reverse genetics

Answer 29

approach that begins with a genotype and proceed to the phenotype by altering the sequence or inhibiting its expression

Question 30

targeted mutagenesis

Answer 30

process where mutations are induced at specific locations

Question 31

site-directed mutations

Answer 31

strategy for mutation where you cut a short sequence of nucleotides with restriction enzymes and replace it with a synthetic oligonucleotide

Question 32

oligonucleotide-directed mutagenesis

Answer 32

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

Question 33

RNAi

Answer 33

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)