Biotechnology Flashcards
1
Q
recombinant DNA tech
A
- allows a DNA fragment from any source to be multiplied by either gene cloning or PCR
- analyzing and altering genes and proteins
- it also provides the reagents necessary for genetic testing like carrier detection, prenatal diagnoses, and gene therapy
- can provide a source of a specific protein in almost unlimited quantities
2
Q
DNA cloning
A
- can produce large amounts of a sequence
- DNA to be cloned present in a small amount in a heterogenous mix of other DNA
- ligate DNA of interest into a piece of nucleic acid referred to as a vector, forming a recombinant vector
3
Q
vectors
A
- usually bacterial or viral plasmids that can be transferred to a host bacterium after insertion of the DNA of interest
- bacteria are then grown in colonies and a colony containing recombinant vector is isolated
- contains a gene for antibiotic resistance so that all other colonies
- the colony left is then grown in large quantities
- bacteria can then be made to express gene of interest or lysed to reisolate the replicated recombinant vectors
- restriction enzymes can release cloned DNA from vector
4
Q
restriction enzymes (restriction endonucleases)
A
- enzymes that recognize specific double-stranded DNA sequences
- these are palindromic, 5’-3’ sequences of the strands are identical (in antiparallel orientation)
- these are isolated from bacteria
- they act as part of a restriction and modification system that protects the bacteria from infection by DNA viruses
- restriction enzyme can cut through the backbones of the double helix
- some of these enzymes produce offset cuts, yielding sticky ends on the fragments; facilitate recombination is a restriction fragment within vector DNA
5
Q
Fragments can be inserted directly into vectors by…
A
choice vectors being cut with the same restriction enzyme.
- DNA vectors contain at least one sequence recognized by restriction enzymes
- vector requires an origin of replication and one gene for antibiotic resistance to allow for recombinant plasmid selection
6
Q
DNA libraries
A
- large collections of known DNA sequences
- to be made, DNA fragments often digested randomly are cloned into vectors and can be utilized for further study
- either genomic DNA or cDNA
7
Q
genomic libraries
A
- contain large fragments of DNA, include both exons and introns
- genes may be split into multiple vectors
- chromosomal DNA
- restriction endonuclease
- promoter and enhancer sequences present
8
Q
cDNA libraries
A
- reverse transcribing processes mRNA
- lacks noncoding regions and only includes genes expressed in the tissues from which mRNA isolated
- aka expression libraries
- cloned genes are complete sequences
- can be used for gene therapy
9
Q
hybridization
A
is the joining of complementary base pair sequences; can be used for DNA-DNA or DNA-RNA recognition
10
Q
PCR
A
- can produce millions of copies of DNA without amplifying DNA in bacteria
- primers complimentary to the DNA w high GC content confer stability flanking the region of interest, nucleotides, and DNA polymerase
- heat
- DNA of interest is denatured, replicated, and then cooled to allow for reannealing of daughter strands w the parent strands
- process repeated
11
Q
gel electrophoresis
A
- separate macros by size and charge
- all DNA molecules are negative
- therefore migrate towards anode of electrochemical cell
- agarose gel and the longer the DNA the slower it will migrate in gel
- performed while using a southern blot
12
Q
southern blot
A
- used to detect presence and quantity of various DNA strands in a sample
- DNA cut by restriction enzymes and then separated by gel electrophoresis
- fragments are then transferred to a membrane retaining separation
- membrane proved with many copies of a single stranded DNA sequence
- probe will bind to complimentary base sequence and form a double-stranded DNA
- probes are labeled with radioisotopes or indicator proteins to indicate presence of desired sequence
13
Q
DNA sequencing
A
- template DNA, primers, an appropriate DNA polymerase, and all 4 deoxyribonucleotide triphosphates
- dideoxyribonucleotides are then added in lower concentrations (ex ddATP) which contain a H at C-3’, rather than a hydroxyl group
- once added polymerase can no longer add to the chain
- after containing many fragments each one will terminate w a modified base
- fragment then separated by size using gel electrophoresis
- last base for each fragment can be read, bc strands separated by size, bases can be easily read in order
14
Q
gene therapy
A
- treats mutated or inactive genes
- transferring a normal copy of gene
- efficient gene delivery vectors must be used for transfer cloned gene into target cell’s DNA
- because viruses naturally infect cells to insert their own genetic material, most gene delivery vectors in use are modified viruses
- a portion of the viral genome is replaced with the cloned gene such that the virus can infect but not complete its replication cycle
15
Q
risk of gene therapy
A
- randomly integrated DNA poses a risk of integrating near and activating a host oncogene
