9-11 DNA technology

Question 1

def restriction endonucease

Answer 1

restriction enzymes that cut double-stranded DNA at specific base sequences (restriction sites) and form smaller DNA fragments

Question 2

def restriction sites

Answer 2

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)

Question 3

def blunt end

Answer 3

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

Question 4

def sticky end

Answer 4

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

Question 5

def ligase

Answer 5

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

Question 6

def linker

Answer 6

short nucleotide base sequences that can be added to blunt ends in order to make them sticky

Question 7

def DNA poly

Answer 7

enzymes which make copies of DNA. DNA polymerases synthesize a single new strand that is complementary to an existing single-stranded DNA template

Question 8

def rev transcriptase

Answer 8

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)

Question 9

def hybridization

Answer 9

refers to techniques involving the annealing of two single strand (of DNA or RNA) of complementary base sequence

Question 10

def probe

Answer 10

commonly used in hybridization techniques to identify or select specific DNA fragments. A probe is a short single-stranded nucleotide of known sequence

Question 11

def southern blot

Answer 11

a hybridization technique used for the identification of DNA fragments through their ability to hybridize with a complementary probe

Question 12

def RFLP

Answer 12

restriction fragment length polymorphism is a technique that exploits variations in homologous DNA sequences. RFLP analysis utilizes Southern blotting

Question 13

def northern blot

Answer 13

hybridization tech for RNA frags

Question 14

def western blot

Answer 14

hybridization tech for proteins

Question 15

def DNA fingerprinting/profiling

Answer 15

a technique used in the identification of individuals by their respective DNA sequences (i.e. DNA profiles) of mini and even smaller microsatellite DNA

Question 16

def vector

Answer 16

vehicle for DNA into cells

Question 17

def transduction

Answer 17

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

Question 18

def transformation

Answer 18

vector uptake w/o phage via prok

Question 19

def transfection

Answer 19

vector uptake w/ phage via euk

Question 20

def recombinant DNA molecule

Answer 20

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

Question 21

def clone

Answer 21

a group of organisms, cells, or molecules all originating from a single individual

Question 22

def genomic library

Answer 22

a collection of DNA fragments from an organism’s entire genome cloned into bacterial colonies

Question 23

def cDNA library

Answer 23

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.)

Question 24

def PCR

Answer 24

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

Question 25

Explain the nomenclature of restriction enzymes. Describe their use and their typical sites of action (restriction sites)

Answer 25

named by the bacteria they are from (Eco from Ecoli) they cut ds DNA into smaller fragments at restr. sites

Question 26

outline southern blot the four applications

Answer 26

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

Question 27

why is cDNA used to express euk protein in bacteria?

Answer 27

since proks do not splice, direct mRNA w/o introns need to be created so cDNA needs to be used

Question 28

explain how cloned DNA is formed

Answer 28

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

Question 29

explain the sanger method of sequencing

Answer 29

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

Question 30

what two diseases can southern blotting/restriction length polymorphisms be tested for?

Answer 30

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

Question 31

what is ASO assay and ie

Answer 31

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

Question 32

what are microarrays?

Answer 32

a large set of ASO bound to a chip that can test for many sequences of random hereditary diseases at once

Question 33

most single gene disorders can be done by direct mutation analysis what four also have alternate testing methodologies?

Answer 33

NF-1, Hemo A, DmD, familial breast cancer can also be ID via linkage analysis also FBC can also be directly sequenced

Question 34

what is somatic cell therapy?

Answer 34

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

Question 35

what is germline therapy?

Answer 35

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

Question 36

what is gene replacement therapy?

Answer 36

a form of somatic cell therapy that works by replacing a mutated gene that causes disease with a healthy gene

Question 37

what is gene therapy for non-inherited diseases?

Answer 37

a form of somatic cell therapy that works by introducing a new gene to help fight a disease

Question 38

what is gene blocking therapy?

Answer 38

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)

Question 39

what is a specific note about using herpes virus as a vector?

Answer 39

are extremely effective at targeting peripheral nerve cells

Question 40

what are the three most common vectors in use?

Answer 40

Adenovirus (23.8%)
Retrovirus (20.5%)
Naked/Plasmid DNA (18.3%)

Question 41

what is the most common disease studied for gene therapy?

Answer 41

cancer

Question 42

what is the general use considerations between vector and non vector usage in gene therapy

Answer 42

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

Question 43

what kind of gene therapy is SCID (x linked and ADA def) treated with?

Answer 43

ex vivo -> cells are extracted, manipulated outside the body, and then re-introduced

Question 44

what kind of gene therapy is Leber’s congenital amaurosis (LCA) treated with?

Answer 44

in vivo -> Treatment occurs within the patient’s body via viral vectors