Genetic Engineering 10.18.12

Question 1

What is DNA sequencing? What is the “chain terminator method”

Answer 1

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.

Question 2

Restriction endonucleases (enzymes)

Answer 2

enzymes that cut double-stranded DNA at very specific recognition sites, based on sequence recognition

Question 3

Where are restriction enzymes found?

Answer 3

bacteria and archea

Question 4

How long are recognitino sequences

Answer 4

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)

Question 5

Some restriction nucleases make a cut with blunt ends while others create sticky ends. what does that mean?

Answer 5

Blunt ends- both strands the same length

Sticky ends- can pair with complementary sequences

Question 6

What is Reverse Transcriptase?

Answer 6

common name for RNA-dependent DNA Polymerase

it uses RNA as a template to synthesize DNA

Opposite to central dogma of mo bio

Question 7

Where do we encounter reverse trancriptase? (3)

Answer 7

1. telomerse
   brings its own RNA along
2. Retrotransposons
   makes use of reverse transcriptase
3. RNA virus
   In HIV virus, RT is the target fr drugs such as AZT, Tenofovir, Didanosine, Sabudine, Efavirenz, and Nevirapine

Question 8

What drugs used for HIV, target the RT?

Answer 8

AZT
Tenofovir
Didanosine
Stavudine
Efavirenze
Nevirapine

Question 9

What can cause DNA to dissociate, melt or denature?

Answer 9

When DNA is heated to >90 ,

high pH (alkylzation) ,

Basic= break

Question 10

Wha happens to pH when DNA is cooled gradually?

Answer 10

pH returns to normal

Complementary strands find each otehr and restore the extensive bp

This is called renaturation or annealing

Question 11

How to you get DNA to renature or anneal

Answer 11

Cool slowly or lower pH (more acidic)

Acid= anneal

Question 12

How can you use DNA’s ability to find its complementary counterpart spontaneously?

Answer 12

used to tag DNA in a sequence-specific manner

Question 13

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

Answer 13

yee

Question 14

What is hybridization?

Answer 14

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

Question 15

What is a polymerase chain reaction?

Answer 15

Important tool in biochemical research

permits the amplification of a single copy or a few copies of DNA to be replicated exponentially

Question 16

What are the necessary components for PCR

Answer 16

1. DNA TEMPLATE to be amplified
2. TWO PRIMERS, one for each strand of DNA . These Can be DNA
3. heat-stable DNA POLYMERASE (taq polymerase usually used )
4. dNTPs (deoxynucletoide triposphahtes) to build the new DNA
5. Divalent cations (Mg2+ commonly used ) and K_

Question 17

List of necessary components for PCR

Answer 17

1. DNA template
2. Two primers
3. DNA polymerase
4. dNTPs
5. Divalent cations and K_

Question 18

Process of PCR

Answer 18

1. Heat mixture >90 degrees –> “DNA melting”
2. Cool mixture to 50-65 degres –>Primers anneal to DNA
3. Heat ot 75-80 degrees then allow polymerase to add nucleotides to primer
4. Each time T cycle is repeated –> amt of DNA doubles

Question 19

How many copies does 10 cycles make? 20 cycles? 30 cycles? of DNA

Answer 19

10- 1024 copies

20- make over a million copies

30- over a billion copies

Question 20

How identical ar two hmans?

Answer 20

99. 99% identical at DNA level (except for ID twins that are 100.00% identical
100. 1% differnce translates to about 3 million sequence differences

Question 21

What are SNPs?

Answer 21

single nucleotide polymorphisms, differences of a single base pair

Question 22

Do SNPs correlate with any difference in PHENOTYPE?

Answer 22

NO!

many of them occur in non-coding regions of genome

Question 23

How come many sgle polymorphisms do not correlate with any difference in phenotype >

Answer 23

1. Many occur in non-coding regions of the genome
2. there is some redundancy somany mutations in coding regiosn do not alter protein sequence
3. many utations in protein sequence dont invovle ciritial parts fo teh protein, so they may not significalty affect fucniton

Question 24

What is an example of a disease wher ea single mutation is sufficient to cause the disease?

Answer 24

Heophilia A

Question 25

What is Hemophilia A and what causes it?

Answer 25

If Factor VIII of coagulation pathwya is deefective, blood does not coagulate efficeintly and there is a tendency to excessive bleeding

Question 26

What is an effective way to dtermine the SNPs responsible for a disease (when more than one SNPs responsible for disease)

Answer 26

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

Question 27

What is Complementary DNA (cDNA)

Answer 27

shortcut to sequencing proteins

Question 28

How is cDNA a shortuct?

Answer 28

bypass having to sequence a eukaryotic gee with introns, exons, and plice sites Isolate mRNA , where introns hav already been spliced out

Question 29

What is the trick to using cDNA

Answer 29

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

Question 30

when is using cDNA useful?

Answer 30

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)

Question 31

How is cDNA useful in proteonomics

Answer 31

By isolating mRNA form different tissues, we can tell which tissues or cell types are producing which proteins under different conditions

Question 32

Creation of cDNA process (from picture)

Answer 32

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

Question 33

what is used as the primer after RNA is degraded with RNAse H

Answer 33

5' end of original mRNA acts as primer

Question 34

What is Cloning?

Answer 34

refers to the process of akign exact copies of DNA (genes) , cells, or organisms

Question 35

What is another method of cloning (besides usng PCR)

Answer 35

Engineering a plasmid

Question 36

What is a plasmid?

Answer 36

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

Question 37

What is anotehr name for plasmid?

Answer 37

Cloning vector

Question 38

how can DNA of interest be inserted inot the plasmid?

Answer 38

restriction nuclease!

Question 39

Wha thappens after plasmid is inserted inot bacterial cell?

Answer 39

microorganism can be grown in clulture plamsmid will replicate and be tranmitted to daugher cells

Question 40

What does Plasmid get to "turn on" the expression of that gene?

Answer 40

a PROMOTER SEQUENCE

Question 41

Why does a plasmid need a replication origin?

Answer 41

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

Question 42

what kind of human proteins have been available via clonign and efrmentation?

Answer 42

Insulin, human growth factor, erythropoitein, and clotting factors

Question 43

What is a transgenic orgnaism?

Answer 43

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

Question 44

What can you learn from mutations that prevent synthesis of a normal protein (KO expts)?

Answer 44

provides insight into the normal function of the gene

Question 45

What happens when defective helicase is engineeered inot mouse on teh righ

Answer 45

transgenic mouse suffers etensive damage to its DNA

Question 46

What is gene replacement?

Answer 46

only mutant gene is active

Question 47

What is gene knockout

Answer 47

no active gene present

Question 48

What is gene additoin

Answer 48

both genes are active (mutatn gene and noral gene x)

Question 49

What is DNA profiling (DNA fingerprinting)

Answer 49

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

Question 50

What part of DNA is used for DNA profiling?

Answer 50

non-coding regions of DNA called STR - short tandem repeats

Question 51

What are short tandem repeats?

Answer 51

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)

Question 52

How do you amplify repeat regions?

Answer 52

PCR

Question 53

What do you use to dermine the number of repeats in each repeat region?

Answer 53

gel electrophoresis

Question 54

How many specific loci are used for forensic purposes

Answer 54

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

Question 55

How can DNA profiling be used in geneological research? What do you use to see paternal lineage? Maternal lineage?

Answer 55

Paternal lineage can be investigated using a set of STR loci on the Y chromosome Maternal lineage can be investigated using mitochondrial DNA

Question 56

What is stopping us from remedying genetic disease by cloning and inserting a fresh new replacement gene?

Answer 56

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?

Question 57

What is first strategic decision in gene therapy?

Answer 57

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

Question 58

Does gene therapy usually focus on germ cell line or somatic?

Answer 58

Somati

Question 59

How are using transposons for gene therapy differnt from using viruses?

Answer 59

Transposons are less efficeint than viruses (less likely to insert) , but they can carry larger genes than many viruses

Question 60

What is an advange of some tranpsosons?

Answer 60

that the have a preferene to insert in non-coding regions of the DNA

Question 61

what are two transposable elemtns that have shown promise as posssible delivery systems for gene therapy?

Answer 61

Sleeping Beauty and piggyBac

Question 62

What does the Sleepy Beauty system consist of?

Answer 62

1. 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

Question 63

What are LIPOLEXES?

Answer 63

Lipid polymers that can form complexes with DNA

Question 64

What are polyplexes?

Answer 64

Cationic polymers that can form complexes with DNA

Question 65

What is the function of LIPOPLEXES and POLYPLEXES?

Answer 65

They can undergo endocytosis, enter cells, and deliver DNA to the nuclus of the ell

Question 66

What variables affect teh efficeinclty of uptake using LIPOPLEXES and POLYPLEXES?

Answer 66

particle size and net charge

Question 67

Are nonparticles (lipoplexes and polyplexes) reliable?

Answer 67

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

Question 68

What is adenovirus? Advantages and disadvantages?

Answer 68

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

Question 69

What is Retrovirus?

Answer 69

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

Question 70

Herpes simplex virus (HSV)

Answer 70

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)