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Genetics > Exam 3: Molecular Genetic Analysis and Biotechnology I > Flashcards

Exam 3: Molecular Genetic Analysis and Biotechnology I Flashcards

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1
Q

recombinant DNA technology (genetic engineering)

A

techniques for locating, isolating, altering, and studying DNA segments. miniaturization and vastly increased computational capacity

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2
Q

the molecular genetics revolution

A

biotechnology; the use of these techniques to develop new products

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3
Q

working at the molecular level

A

it is now possible to sequence a single molecule of DNA

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4
Q

first step of molecular genetics

A

isolate DNA segment or gene from remaining DNA

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5
Q

cutting and joining DNA fragments via

A

restriction enzymes

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6
Q

locating DNA fragments via

A

with southern blotting and probes

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7
Q

restriction enzymes

A

(isolation) recognizing and cutting DNA at specific nucleotide sequences; palindromic sequence

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8
Q

type II restriction enzyme

A

cut DNA at defined positions close to or w.in their recognition sequences. most useful restriction enzymes

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9
Q

proks use restriction enzymes to

A

ward off viral parasites. by adding methyl groups to their own recognition sequence to protect itself from being digested and only cutting up the DNA of the invader

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10
Q

cohesive ends

A

fragments with short, single-stranded overhanging ends

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11
Q

blunt ends

A

even-length ends from both single strands

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12
Q

restriction enzymes make _ cuts in DNA producing _

A

double-stranded cuts producing cohesive, or sticky, ends

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13
Q

promoter

A

a promoter is a region of DNA where transcription of a gene is initiated. Promoters are a vital component of expression vectors because they control the binding of RNA polymerase to DNA (must be close to a gene)

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14
Q

enhancer

A

Enhancer sequences are regulatory DNA sequences that, when bound by specific proteins called transcription factors, enhance the transcription of an associated gene (can be far away from a gene)

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15
Q

start codon

A

AUG, marks the beginning of a protein and also encodes the amino acid methionine
Codons in an mRNA are read during translation, beginning with a start codon

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16
Q

transcription initiation site

A

The transcription start site is the location where transcription starts at the 5’-end of a gene sequence.

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17
Q

gel electrophoresis

A

DNA has a negative charge so it will migrate toward the + electrode. separation is based on fragment length

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18
Q

gel electrophoresis: DNA is usu cut with _ or is the product of _

A

cut w/ restriction enzymes or is the product of PCR

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19
Q

gel electrophoresis: the gel is soaked in _ which sticks to _

A

ethidium bromide (EB) which sticks to dsDNA and fluoresces when exposed to UV light

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20
Q

probe

A

DNA or RNA with a base seq complementary to a seq in the gene of interest (antisense), and w/ a radioactive or chemiluminescent molecule attached allows visualization

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21
Q

southern blotting and hybridization with radioactive or fluorescent probe can locate _

A

a few specific fragments in a large pool of DNA

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22
Q

RPE64/65; Leber congenital amaurosis

A

gene that encodes protein involved in pigment formation in the eye; gene therapy to treat genetic blindness that optimizes the promoter and a codon-optimized gene all to stabilize modified promoter with new gene for insertion to treat (allow stable expression of gene)

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23
Q

purpose of cloning genes once identified

A

need to amplify for studies

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24
Q

gene cloning

A

amplifying a specific piece of DNA via a bacterial cell

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25
Q

cloning vector

A

a replicating DNA molecule incorporating a foreign DNA sequence to be introduced into a cell

26
Q

cloning genes: 3 aspects

A
  1. plasmid vectors
  2. transformation of host cells with plasmids
  3. screening cells for recombinant plasmids
27
Q

plasmid vectors have: (3)

A
  1. plasmids: circular DNA molecules from bacteria
  2. insert foreign DNA into plasmid using restriction enzymes
  3. linkers: synthetic DNA fragments containing restriction sites
28
Q

screening cells for recombinant plasmids (cloning genes)

A

selectable markers (eg antibiotic resistance genes) are used to confirm whether the cells have been transformed or not

29
Q

an idealized cloning vector has an _ (3 things)

A

origin or replication, one or more selectable markers, and one or more unique restriction sites

30
Q

first, a cloning vector must contain an origin of replication recognized in the host cell so

A

that it is replicated along with the DNA that it carries

31
Q

second, a cloning vector should carry selectable markers bc

A

it will have traits that enable cells containing the vector to be selected or identified

32
Q

third, a cloning vector needs a single cleavage site for each of

A

one or more restriction enzyme used

33
Q

lacZ gene encodes

A

the protein beta-galactosidase, which will break down X-gal to form a blue precipitate (screen bacteria containing recombinant plasmids)

34
Q

bacterial colonies which contain a functional lacZ gene will be _ when grown on media containing X-gal

A

blue (visual marker)

35
Q

when lacZ is interrupted by an inserted DNA fragment, the colonies will be _

A

white

36
Q

t/f: a foreign DNA fragment can be inserted into a plasmid with the use of restriction enzymes

A

true

37
Q

yeast artificial chromosomes (YACs) 100-1000kb replaced by BACs bc of

A

instability issues

38
Q

to ensure transcription and translation, a _ gene may be inserted into an expression vector

A

foreign gene

39
Q

expression vectors contain _ sequences that allow inserted DNA to be transcribed and translated. they also include sequences that _ the desired gene

A

operon sequences; sequences that regulate (turn on or turn off) the desired gene

40
Q

the _ plasmid can be used to transfer genes into plants

A

Ti plasmid

41
Q

t/f: molecular genetic techniques have been used to create genetically modified crops

A

true; genetically engineered corn now constitutes 88% of all corn grown in the US

42
Q

Round-Up

A

glyphosate (herbicide) inhibits an essential plant enzyme, EPSPS, involved in the synthesis of 3 aromatic aas: tyrosine, tryptophan, and phenylalanine. some micro-organisms have a version of EPSPS that is resistant to glyphosate. furthermore, causes cancer as does not only work in plants as thought

43
Q

the pcr rxn

A

there are 5 chemical components needed for a PCR rxn: a DNA template, DNA polymerase enzyme, primers, nucleotides and reaction buffer

44
Q

taq polymerase

A

DNA polymerase enzyme that is stable at boiling temperatures (isolated from bacteria that survive in Old Faithful)

45
Q

reverse-transcription PCR

A

in RT-PCR, the RNA template is first converted into a complementary DNA (cDNA) using a reverse transcriptase. the cDNA is then used as a template for exponential amplification using PCR

46
Q

the use of thermostable polymerases made PCR _

A

automatable

47
Q

RT-PCR uses reverse transcriptases derived from _

A

retroviruses to convert RNA to DNA followed by PCR

48
Q

_ won the 1993 Nobel prize for his work perfecting the polymerase chain rxn

A

kerry mullis

49
Q

quantitative real-time PCR

A

quantitatively determining the amount of DNA amplified as the reaction proceeds (sybr green)

50
Q

sybr green

A

emission of the fluorescence when bound to double stranded DNA

51
Q

multiplexing real-time PCR, probes with different spectral wavelengths can be use to allow _

A

the quantification of multiple targets in a single reaction

52
Q

strengths of PCR (5)

A
  • detect virus presence in blood samples
  • identify genetic variation
  • isolate DNA from ancient sources
  • crime scenes (amplify small amts of DNA)
  • introduce new seqs into a fragment of DNA
53
Q

limitations of PCR (4)

A
  • prior knowledge of sequences to construct primers
  • contamination
  • taq does not proofread
  • size of fragment that can be amplified is less than 2000bp
54
Q

dna library

A

collection of clones containing all the DNA fragments from one source; contains all of the DNA seqs found in an organism’s genome

55
Q

creating a genomic DNA library

A

take DNA from genome, cut it up, put in a vector to amplify, and maintaining clones for storage and future amplification

56
Q

cDNA libraries

A

consisting only of those DNA sequences that are transcribed into mRNA

57
Q

screening DNA libraries (2)

A
  1. plating clones of the library

2. probing plated colonies or plaques (hybridizing to complementary DNA)

58
Q

in situ hybridization (2)

A
  • DNA probes used to determine the chromosomal location and to visualize a gene while it is in a cell
  • FISH; light is applied to see fluoresce
59
Q

chromosome walking

A

chromosome walking is a technique used to clone a gene (eg disease gene) from its known closest markers (eg known gene) and hence is used in moderate modifications in cloning and sequencing projects

60
Q

chromosome jumping

A

chromosome jumping allows the use of one point on a chromosome as a starting point for exploring another potentially distant point on the same chromosome without cloning the intervening sequences as in chromosome walking

61
Q

identification of the cystic fibrosis gene: chromosome walking and jumping

A

Dr Francis Collins et all utilised the technique of ‘chromosome jumping’. This allowed them to move over large distances of the long arm of chromosome 7 relatively quickly. after every jump the position of each clone with respect to the gene defective in cystic fibrosis (CF) was checked using genetic and physical mapping methods.

62
Q

mummy Ata

A

whole genome sequencing showed that this mummy was a miscarried fetus due to many mutations, severely in genes needed for pathways in bone development and ossification

Genetics (37 decks)

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