9-11 DNA technology Flashcards
def restriction endonucease
restriction enzymes that cut double-stranded DNA at specific base sequences (restriction sites) and form smaller DNA fragments
def restriction sites
a specific sequence of bases, usually 4-6 base pairs in length, which is cleaved by a restriction enzyme (e.g. restriction endonucleases). Many restriction sites are palindromic (i.e. both strands have the same base sequence when read in the 5’ to 3’ direction)
def blunt end
DNA fragment that contains no overhang at either the 5’ or 3’ end and consequently has no DNA bases available for hybridization to other DNA fragments. Blunt ends are formed when a restriction enzyme cleaves a DNA molecule in a straight-through fashion
def sticky end
end of a DNA double helix at which a few unpaired nucleotides of one strand extend beyond the other. Sticky ends are created when a restriction enzyme cleaves a DNA molecule in a staggered fashion, producing an overhang
def ligase
enzymes which join together fragments of DNA. In vivo they repair single-stranded breaks, while in vitro they are used to join fragments of double-stranded DNA. The joining together of DNA molecules is referred to as ligation
def linker
short nucleotide base sequences that can be added to blunt ends in order to make them sticky
def DNA poly
enzymes which make copies of DNA. DNA polymerases synthesize a single new strand that is complementary to an existing single-stranded DNA template
def rev transcriptase
an enzyme that copies RNA onto DNA. Reverse transcriptase synthesizes a DNA strand complementary to an single-stranded RNA template (i.e. it performs the reverse of transcription)
def hybridization
refers to techniques involving the annealing of two single strand (of DNA or RNA) of complementary base sequence
def probe
commonly used in hybridization techniques to identify or select specific DNA fragments. A probe is a short single-stranded nucleotide of known sequence
def southern blot
a hybridization technique used for the identification of DNA fragments through their ability to hybridize with a complementary probe
def RFLP
restriction fragment length polymorphism is a technique that exploits variations in homologous DNA sequences. RFLP analysis utilizes Southern blotting
def northern blot
hybridization tech for RNA frags
def western blot
hybridization tech for proteins
def DNA fingerprinting/profiling
a technique used in the identification of individuals by their respective DNA sequences (i.e. DNA profiles) of mini and even smaller microsatellite DNA
def vector
vehicle for DNA into cells
def transduction
vector uptake w/ phage via prok
generalized = random set that can lead to DNA not incorp, DNA recirc to plasmid, homologue DNA replacing original
specialized = excision of some of bact DNA around the splice site for phage DNA
def transformation
vector uptake w/o phage via prok
def transfection
vector uptake w/ phage via euk
def recombinant DNA molecule
a form of artificial DNA that is created by combining two or more sequences that would not normally occur together in nature ie. insulin gene on a plasmid vector
def clone
a group of organisms, cells, or molecules all originating from a single individual
def genomic library
a collection of DNA fragments from an organism’s entire genome cloned into bacterial colonies
def cDNA library
a collection of cDNA fragments produced from all the mRNA present in a particular tissue types (e.g. liver cDNA library, pancreatic cDNA library, etc.)
def PCR
polymerase chain reaction is a technique which amplifies a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence
Explain the nomenclature of restriction enzymes. Describe their use and their typical sites of action (restriction sites)
named by the bacteria they are from (Eco from Ecoli) they cut ds DNA into smaller fragments at restr. sites
outline southern blot the four applications
south - DNA, restricted and fragments separated by size via gel electrophoresis
longer length shorter dist traveled
results xfered onto nitrocell and probed via autoradiography
used for 1ID genes 2Dx diseases 3ID Carriers 4DNA fingerprinting
why is cDNA used to express euk protein in bacteria?
since proks do not splice, direct mRNA w/o introns need to be created so cDNA needs to be used
explain how cloned DNA is formed
recombinant DNA via plasmid vector and target gene is created by cleaving plasmid at one site with ends that fit original DNA and it is ligated then incorporated to bacteria which will then prolif and plasmids are isolated then pure target gene that are cloned can be cleaved out
explain the sanger method of sequencing
aka chain termination/dideoxy method in which DNA is exposed random primers, nucleotides and phosphorylated nucleotides
hybridization then gel electrophoresis will lead to manual sequencing via pc screen and lowest fragment to higher fragment (down to up) in reading the DNA sequence from 3’ to 5’ end
what two diseases can southern blotting/restriction length polymorphisms be tested for?
sickle cell disease - MstII restriction site on beta globin gene is absent in pts w/ sickle cell –> longer fragments (1.3kb is Dx, 1.1 is normal, both is carrier)
myotonic dystrophy causing triplet expansion can be out of range for RFLP assay so nothing shows up –> may need a expanded primer hybridization step
what is ASO assay and ie
ASO is a short sequence (oligonucleotide) probe that simply binds to the sequence in question, sequence needed must be short and known
commonly tested are for sickle cell and cystic fibrosis
only need to amplify the two sequences of interest
what are microarrays?
a large set of ASO bound to a chip that can test for many sequences of random hereditary diseases at once
most single gene disorders can be done by direct mutation analysis what four also have alternate testing methodologies?
NF-1, Hemo A, DmD, familial breast cancer can also be ID via linkage analysis also FBC can also be directly sequenced
what is somatic cell therapy?
therapeutic genes are transferred into the somatic cells, or body, of a patient. Any modifications and effects will be restricted to the individual patient only, and will not be inherited by the patient’s offspring or later generations
what is germline therapy?
germ cells (i.e. sperm or eggs) are modified by the introduction of functional genes, which are integrated into their genomes. This would allow the therapy to be heritable and passed on to later generations
what is gene replacement therapy?
a form of somatic cell therapy that works by replacing a mutated gene that causes disease with a healthy gene
what is gene therapy for non-inherited diseases?
a form of somatic cell therapy that works by introducing a new gene to help fight a disease
what is gene blocking therapy?
a form of somatic cell therapy that works by inactivating a mutated gene that is functioning improperly (e.g. antisense therapy, ribozyme therapy, RNA interference)
what is a specific note about using herpes virus as a vector?
are extremely effective at targeting peripheral nerve cells
what are the three most common vectors in use?
Adenovirus (23.8%)
Retrovirus (20.5%)
Naked/Plasmid DNA (18.3%)
what is the most common disease studied for gene therapy?
cancer
what is the general use considerations between vector and non vector usage in gene therapy
non-viral vectors are better suited for ex vivo (i.e. outside of an organism) gene therapy than for in vivo (i.e. inside a living organism) gene therapy
what kind of gene therapy is SCID (x linked and ADA def) treated with?
ex vivo -> cells are extracted, manipulated outside the body, and then re-introduced
what kind of gene therapy is Leber’s congenital amaurosis (LCA) treated with?
in vivo -> Treatment occurs within the patient’s body via viral vectors
