Genetic Engineering 10.18.12 Flashcards
What is DNA sequencing? What is the “chain terminator method”
uses 2’, 3’ -dideoxribose, which can be incorporated into a growing DNA chain by DNA polyerase (which lacks a 3’OH and prevents further elongation of the chain.
Restriction endonucleases (enzymes)
enzymes that cut double-stranded DNA at very specific recognition sites, based on sequence recognition
Where are restriction enzymes found?
bacteria and archea
How long are recognitino sequences
Typically 4-8 nucleotides in length
enzyme that recognizes a 4 nucleotide sequence should, cut once every 256 (4x4x4x4) bp (or cut DNA into fragmetns iwth avg size of 56 bp)
Some restriction nucleases make a cut with blunt ends while others create sticky ends. what does that mean?
Blunt ends- both strands the same length
Sticky ends- can pair with complementary sequences
What is Reverse Transcriptase?
common name for RNA-dependent DNA Polymerase
it uses RNA as a template to synthesize DNA
Opposite to central dogma of mo bio
Where do we encounter reverse trancriptase? (3)
- telomerse
brings its own RNA along - Retrotransposons
makes use of reverse transcriptase - RNA virus
In HIV virus, RT is the target fr drugs such as AZT, Tenofovir, Didanosine, Sabudine, Efavirenz, and Nevirapine
What drugs used for HIV, target the RT?
AZT Tenofovir Didanosine Stavudine Efavirenze Nevirapine
What can cause DNA to dissociate, melt or denature?
When DNA is heated to >90 ,
high pH (alkylzation) ,
Basic= break
Wha happens to pH when DNA is cooled gradually?
pH returns to normal
Complementary strands find each otehr and restore the extensive bp
This is called renaturation or annealing
How to you get DNA to renature or anneal
Cool slowly or lower pH (more acidic)
Acid= anneal
How can you use DNA’s ability to find its complementary counterpart spontaneously?
used to tag DNA in a sequence-specific manner
If DNA samples are separated by electrophoresis and transferred to nitrocellulose, DNA with a specific sequence and radioactive label can pair with complementary DNA on the nitrocellulose (hybridization), allowing deterciton of DNA with specific protperties
yee
What is hybridization?
when DNA with a specific sequence and a readioactive label can pair with complemeentary DNA on the nitrocellulase, allwing detection of DNA with specific porperties
What is a polymerase chain reaction?
Important tool in biochemical research
permits the amplification of a single copy or a few copies of DNA to be replicated exponentially
What are the necessary components for PCR
- DNA TEMPLATE to be amplified
- TWO PRIMERS, one for each strand of DNA . These Can be DNA
- heat-stable DNA POLYMERASE (taq polymerase usually used )
- dNTPs (deoxynucletoide triposphahtes) to build the new DNA
- Divalent cations (Mg2+ commonly used ) and K_
List of necessary components for PCR
- DNA template
- Two primers
- DNA polymerase
- dNTPs
- Divalent cations and K_
Process of PCR
- Heat mixture >90 degrees –> “DNA melting”
- Cool mixture to 50-65 degres –>Primers anneal to DNA
- Heat ot 75-80 degrees then allow polymerase to add nucleotides to primer
- Each time T cycle is repeated –> amt of DNA doubles
How many copies does 10 cycles make? 20 cycles? 30 cycles? of DNA
10- 1024 copies
20- make over a million copies
30- over a billion copies
How identical ar two hmans?
- 99% identical at DNA level (except for ID twins that are 100.00% identical
- 1% differnce translates to about 3 million sequence differences
What are SNPs?
single nucleotide polymorphisms, differences of a single base pair
Do SNPs correlate with any difference in PHENOTYPE?
NO!
many of them occur in non-coding regions of genome
How come many sgle polymorphisms do not correlate with any difference in phenotype >
- Many occur in non-coding regions of the genome
- there is some redundancy somany mutations in coding regiosn do not alter protein sequence
- many utations in protein sequence dont invovle ciritial parts fo teh protein, so they may not significalty affect fucniton
What is an example of a disease wher ea single mutation is sufficient to cause the disease?
Heophilia A
What is Hemophilia A and what causes it?
If Factor VIII of coagulation pathwya is deefective, blood does not coagulate efficeintly and there is a tendency to excessive bleeding
What is an effective way to dtermine the SNPs responsible for a disease (when more than one SNPs responsible for disease)
Determine SNPs for a gorup of people witha particular disease and compare with SNPs of a groupw ithout eh disease , and to look for SNPs that coorrelate
What is Complementary DNA (cDNA)
shortcut to sequencing proteins
How is cDNA a shortuct?
bypass having to sequence a eukaryotic gee with introns, exons, and plice sites
Isolate mRNA , where introns hav already been spliced out
What is the trick to using cDNA
Isolate mRNA wher eintrons have already been spliced out
Use Reverse Transriptase to make a DNA copy
use PCR to amplify the number of copies
use Automated sequences to get the DNA seqeucnes
based on Genetic Code, we can predict the amino acid seuqence
when is using cDNA useful?
if you want to express a eukaryotic protein in a prokaryotic organism
remember: rpokaryotes cant process our the introns
cDNA is also useful in proteonomics (study of proteins)
How is cDNA useful in proteonomics
By isolating mRNA form different tissues, we can tell which tissues or cell types are producing which proteins under different conditions
Creation of cDNA process (from picture)
Brain –> lyse cells and purify mRNA –> hybridize with poly (T) primer –> make DNA copy with Reverse Transcriptase –> degrade RNA with RNAse H –> synthesize a complementary DNA strand using a DNA polymerase; 5’ end of original mRNA actss as primer
what is used as the primer after RNA is degraded with RNAse H
5’ end of original mRNA acts as primer
What is Cloning?
refers to the process of akign exact copies of DNA (genes) , cells, or organisms
What is another method of cloning (besides usng PCR)
Engineering a plasmid
What is a plasmid?
piece of extra-chromosomal circular DNA)
insert it into a bacterium
DNA of interest can be inserted inot teh plasmid (also called a cloning vector) by means of restriction nuclease
Once plasmid is inserted into bacterial cell ,the microorganism can be grown in culture; the plasmid will replicate and be transmitted to daughter cells
What is anotehr name for plasmid?
Cloning vector
how can DNA of interest be inserted inot the plasmid?
restriction nuclease!
Wha thappens after plasmid is inserted inot bacterial cell?
microorganism can be grown in clulture
plamsmid will replicate and be tranmitted to daugher cells
What does Plasmid get to “turn on” the expression of that gene?
a PROMOTER SEQUENCE
Why does a plasmid need a replication origin?
so that it will be copied when teh cell replicates
once this is accomplished, host scell will usually express the gene, making mRNA whihc, in turn, leds to synthesis of corresponding protein
what kind of human proteins have been available via clonign and efrmentation?
Insulin, human growth factor, erythropoitein, and clotting factors
What is a transgenic orgnaism?
When you ngineer the cloned gene into whole eukaryotic organisms (mice for example)
Single point mutations can be introduced to explore protein structure and function
What can you learn from mutations that prevent synthesis of a normal protein (KO expts)?
provides insight into the normal function of the gene
What happens when defective helicase is engineeered inot mouse on teh righ
transgenic mouse suffers etensive damage to its DNA
What is gene replacement?
only mutant gene is active
What is gene knockout
no active gene present
What is gene additoin
both genes are active (mutatn gene and noral gene x)
What is DNA profiling (DNA fingerprinting)
useful in forensic science to disntingish DNA from different individuals, or to estimate the probablity that a DNA smaple came from a particular indiviual
or est. how related two peopel are
What part of DNA is used for DNA profiling?
non-coding regions of DNA called STR - short tandem repeats
What are short tandem repeats?
non-coding regions of DNA
ie CACACA or GTGTG or CATGCATG….
part of a larger classs of repetitive DNA called VNTR (variable number tandem repeats)
How do you amplify repeat regions?
PCR
What do you use to dermine the number of repeats in each repeat region?
gel electrophoresis
How many specific loci are used for forensic purposes
set of 13 specific loci (specific locations on specific chromosomes)
Loci are independtly assorted, so it is possible to calculate the probabliyt tha ny two randomly chosen indivduals would have the same DNA prfile is 1x10^18
How can DNA profiling be used in geneological research?
What do you use to see paternal lineage? Maternal lineage?
Paternal lineage can be investigated using a set of STR loci on the Y chromosome
Maternal lineage can be investigated using mitochondrial DNA
What is stopping us from remedying genetic disease by cloning and inserting a fresh new replacement gene?
Primarily, it’s the installation process that is most difficult
How do you relaibly deliver gene to all the cells that need it?
How do you get it incorporated into the genome of each ell?
How do you make sure you don’t overdose? (get too many copies inserted per cell?
How do you make sure it doesn’t insert ina way that would damage other improtant genes?
What is first strategic decision in gene therapy?
whether to make change in germline cell (egg o sperm) or somatic cell?
Repairing a gene in a germline cell would pre-emtp expressino of a genetic defect in one’s offspring and repair would be passed on to future genearsion
Gene repair in somatic cells line would be useful in treating someone who already has a genetic defect
Does gene therapy usually focus on germ cell line or somatic?
Somati
How are using transposons for gene therapy differnt from using viruses?
Transposons are less efficeint than viruses (less likely to insert) , but they can carry larger genes than many viruses
What is an advange of some tranpsosons?
that the have a preferene to insert in non-coding regions of the DNA
what are two transposable elemtns that have shown promise as posssible delivery systems for gene therapy?
Sleeping Beauty and piggyBac
What does the Sleepy Beauty system consist of?
- Transposon vector (top) contains gene to be inserted, flanked on both ends by Sleeping Beauty terminal invered rpeates
The other component is the Transposase Expression vectro (bottom) ,which contains teh tranposase gene witha astrong promoter and a poly-A Tail
What are LIPOLEXES?
Lipid polymers that can form complexes with DNA
What are polyplexes?
Cationic polymers that can form complexes with DNA
What is the function of LIPOPLEXES and POLYPLEXES?
They can undergo endocytosis, enter cells, and deliver DNA to the nuclus of the ell
What variables affect teh efficeinclty of uptake using LIPOPLEXES and POLYPLEXES?
particle size and net charge
Are nonparticles (lipoplexes and polyplexes) reliable?
No. there are numerous ways DNA may bget sidetracked or destroyed along teh way
for example , if endocytosed material ends up in lyososmes, DNA is lkey to be destroyed by nucleases
once DA is liberated inside the cell, it must ifnd its way to the nuclues’ one way to accomplish this would be to assocaite it with a protein having a nulcear localiation sequence
What is adenovirus?
Advantages and disadvantages?
double-stranded DNA virus
Main advantage is that its DNA is not incorporated inot teh host cell DNA –it is a separte ,free piece of DNA
It will NOT accidentally insert int some other improtnat gene
Disadvantage- newly introduced DNA will not be transmitted t daughter cells when cell replicates
What is Retrovirus?
carry genetic ino as ss RNA
Hvae a RT whcih makes a NA copy, and an integrase which inserts that DNA into host cell’s genome
New gene will be inherited by daughter cells ,but it is not yet possible to control where new gene is inserted
Herpes simplex virus (HSV)
delivery vector represens an example of tissue targeting.
HSV can infect neurons,so modified HSV has been used to target the NS. It has been used to target some gliomas (tumors derived from nerve cells)
