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CMCB Fall > Genetic Engineering 10.18.12 > Flashcards

Genetic Engineering 10.18.12 Flashcards

1
Q

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

A

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.

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2
Q

Restriction endonucleases (enzymes)

A

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

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3
Q

Where are restriction enzymes found?

A

bacteria and archea

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4
Q

How long are recognitino sequences

A

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)

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5
Q

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

A

Blunt ends- both strands the same length

Sticky ends- can pair with complementary sequences

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6
Q

What is Reverse Transcriptase?

A

common name for RNA-dependent DNA Polymerase

it uses RNA as a template to synthesize DNA

Opposite to central dogma of mo bio

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7
Q

Where do we encounter reverse trancriptase? (3)

A
  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
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8
Q

What drugs used for HIV, target the RT?

A 
AZT
Tenofovir
Didanosine
Stavudine
Efavirenze
Nevirapine
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9
Q

What can cause DNA to dissociate, melt or denature?

A

When DNA is heated to >90 ,

high pH (alkylzation) ,

Basic= break

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10
Q

Wha happens to pH when DNA is cooled gradually?

A

pH returns to normal

Complementary strands find each otehr and restore the extensive bp

This is called renaturation or annealing

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11
Q

How to you get DNA to renature or anneal

A

Cool slowly or lower pH (more acidic)

Acid= anneal

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12
Q

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

A

used to tag DNA in a sequence-specific manner

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13
Q

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

A

yee

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14
Q

What is hybridization?

A

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

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15
Q

What is a polymerase chain reaction?

A

Important tool in biochemical research

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

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16
Q

What are the necessary components for PCR

A
  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_
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17
Q

List of necessary components for PCR

A
  1. DNA template
  2. Two primers
  3. DNA polymerase
  4. dNTPs
  5. Divalent cations and K_
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18
Q

Process of PCR

A
  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
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19
Q

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

A

10- 1024 copies

20- make over a million copies

30- over a billion copies

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20
Q

How identical ar two hmans?

A
  1. 99% identical at DNA level (except for ID twins that are 100.00% identical
  2. 1% differnce translates to about 3 million sequence differences
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21
Q

What are SNPs?

A

single nucleotide polymorphisms, differences of a single base pair

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22
Q

Do SNPs correlate with any difference in PHENOTYPE?

A

NO!

many of them occur in non-coding regions of genome

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23
Q

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

A
  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
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24
Q

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

A

Heophilia A

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25
Q

What is Hemophilia A and what causes it?

A

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

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26
Q

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

A

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

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27
Q

What is Complementary DNA (cDNA)

A

shortcut to sequencing proteins

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28
Q

How is cDNA a shortuct?

A

bypass having to sequence a eukaryotic gee with introns, exons, and plice sites

Isolate mRNA , where introns hav already been spliced out

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29
Q

What is the trick to using cDNA

A

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

30
Q

when is using cDNA useful?

A

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)

31
Q

How is cDNA useful in proteonomics

A

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

32
Q

Creation of cDNA process (from picture)

A

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

33
Q

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

A

5’ end of original mRNA acts as primer

34
Q

What is Cloning?

A

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

35
Q

What is another method of cloning (besides usng PCR)

A

Engineering a plasmid

36
Q

What is a plasmid?

A

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

37
Q

What is anotehr name for plasmid?

A

Cloning vector

38
Q

how can DNA of interest be inserted inot the plasmid?

A

restriction nuclease!

39
Q

Wha thappens after plasmid is inserted inot bacterial cell?

A

microorganism can be grown in clulture

plamsmid will replicate and be tranmitted to daugher cells

40
Q

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

A

a PROMOTER SEQUENCE

41
Q

Why does a plasmid need a replication origin?

A

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

42
Q

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

A

Insulin, human growth factor, erythropoitein, and clotting factors

43
Q

What is a transgenic orgnaism?

A

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

44
Q

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

A

provides insight into the normal function of the gene

45
Q

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

A

transgenic mouse suffers etensive damage to its DNA

46
Q

What is gene replacement?

A

only mutant gene is active

47
Q

What is gene knockout

A

no active gene present

48
Q

What is gene additoin

A

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

49
Q

What is DNA profiling (DNA fingerprinting)

A

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

50
Q

What part of DNA is used for DNA profiling?

A

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

51
Q

What are short tandem repeats?

A

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)

52
Q

How do you amplify repeat regions?

A

PCR

53
Q

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

A

gel electrophoresis

54
Q

How many specific loci are used for forensic purposes

A

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

55
Q

How can DNA profiling be used in geneological research?

What do you use to see paternal lineage? Maternal lineage?

A

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

Maternal lineage can be investigated using mitochondrial DNA

56
Q

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

A

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?

57
Q

What is first strategic decision in gene therapy?

A

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

58
Q

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

A

Somati

59
Q

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

A

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

60
Q

What is an advange of some tranpsosons?

A

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

61
Q

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

A

Sleeping Beauty and piggyBac

62
Q

What does the Sleepy Beauty system consist of?

A
  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

63
Q

What are LIPOLEXES?

A

Lipid polymers that can form complexes with DNA

64
Q

What are polyplexes?

A

Cationic polymers that can form complexes with DNA

65
Q

What is the function of LIPOPLEXES and POLYPLEXES?

A

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

66
Q

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

A

particle size and net charge

67
Q

Are nonparticles (lipoplexes and polyplexes) reliable?

A

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

68
Q

What is adenovirus?

Advantages and disadvantages?

A

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

69
Q

What is Retrovirus?

A

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

70
Q

Herpes simplex virus (HSV)

A

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)

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