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Genetics > Lecture 14: Genetics Technologies > Flashcards

Lecture 14: Genetics Technologies Flashcards

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1
Q

how are restriction enzymes produced?

A

by bacteria as a defense mechanism against infection by viruses

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2
Q

how many restriction enzymes have been identified?

A
  • 3500

- 150 are commonly used

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3
Q

recognition sequence

A

a specific nucleotide sequence recognized by restriction enzymes, cut by endonuclease activity

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4
Q

what are the purposes of restriction enzymes?

A
  1. making recombinant DNA and appraising success for research, medicine, and agriculture
  2. DNA profile anlysis for disease diagnosis, paternity/family relationship testing, and forensics
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5
Q

what are the protruding ends that were cut by restriction enzymes called?

A

sticky ends

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6
Q

what is the purpose of restriction fragments?

A

making recombinant DNA

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7
Q

how is recombinant DNA made?

A

when you cut two separate molecules of DNA with the same restriction enzyme, the fragments will have matching sticky ends

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8
Q

RFLPs

A
  • restriction fragment length polymorphism
  • marker
  • presence of absence of restriction sites to be digested by restriction enzymes
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9
Q

VNTR

A
  • variable number of tandem repeats
  • marker
  • in the human genome, there exists short repetitive portions of DNA. the lengths of these repetitive sequences will vary from individual to individual, producing unique banding pattern
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10
Q

SNPs

A
  • single nucleotides polymorphisms
  • marker
  • rare substitution differences, about .001 frequency in humans (1 SNP every 1000bp). SNPs often constitute different alleles
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11
Q

PCR sites

A
  • marker

- presence or absence of priming sites, the ability for primers to anneal to an individual’s DNA

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12
Q

what are the steps for RFLP?

A
  • DNA extraction
  • PCR with labeled primers
  • digestion with restriction enzymes
  • electrophoresis separation
  • laser detection
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13
Q

vectors

A

carrier DNA molecules that transfer and help replicate inserted DNA fragments

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14
Q

how do vectors vary?

A
  • differ in the hosts they are able to enter

- size of inserts they can carry

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15
Q

what should vectors include?

A
  • should contain several restriction enzyme cleavage sites that allow insertion of DNA fragments to be clones
  • they must be able to independently replicate themselves and any DNA fragment they carry once they are inside a host cell
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16
Q

selectable marker genes

A

to distinguish host cells that have taken up vectors from those have not, usually an antibiotic resistance gene or a color changing gene

17
Q

plasmid

A
  • vector

- 10-20 kb of target DNA

18
Q

bacteriophage

A
  • vector

- lambda can carry a 15kb insert

19
Q

cosmids

A
  • vector

- a plasmid + lambda DNA hybrid, 50kb inserts

20
Q

bacterial artificial chromosomes (BACs)

A
  • vector

- F plasmids that can accept 150-300kb inserts

21
Q

yeast artificial chromosomes (YACs)

A
  • vector

- can accept big inserts, most useful in eukaryotes (~1M)

22
Q

polymerase chain reaction (PCR)

A
  • rapid method of DNA cloning used to generate millions of copies in short time
  • copies specific DNA sequence via in vitro reactions; can amplify target DNA sequences present in very small quantities
23
Q

what are the components of PCR?

A
  • two primers - both primers anneal to denatured DNA
  • dsDNA to be cloned is put in a tube with DNA polymerase, Mg 2+ and dNTPs
  • complementary strands are synthesized by heat-stable DNA polymerase
24
Q

what are the three steps of PCR?

A
  • denaturation
  • primer annealing
  • extention
  • steps are repeated over and over using a thermocycler to amplify DNA exponentially
25
Q

what are the limitations of PCR?

A
  • need for target DNA sequence information
  • minor contamination from other sources can cause problems
  • short size and limiting amounts
  • primers have self-annealing regions within each primer preventing its annealing to target DNA
26
Q

reverse transcription PCR (RT-PCR)

A
  • methodology for studying gene expression

- reverse transcriptase is used to generate ds-cDNA

27
Q

quantitative real-time PCR (qPCR)

A

-real time PCR allows researchers to quantify amplification reactions as they occur in real time

28
Q

DNA ligase

A
  • DNA fragments will seal phosphodiester backbone

- joints restriction fragments covalently to produce intact DNA molecules

29
Q

multiple cloning sites

A
  • restriction sites for commonly used restriction enzymes

- allow scientists to clone a range of different fragments

30
Q

what are the applications of PCR?

A
  • most widely used technique in genetics and molecular biology
  • allows for screening of mutations involved in genetic disorders
  • location and nature of mutation can be determined quickly
31
Q

reverse transcription PCR (RT-PCR)

A
  • methodology for studying gene expression (mRNA production by cells or tissues)
  • reverse transcriptase is used to generate ds-cDNA
32
Q

quantitative real-time PCR (qPCR)

A

-real time PCR allows researchers to quantify amplification reactions as they occur in real time

33
Q

vectors

A
  • carrier DNA molecules
  • can replicate cloned DNA fragments in host cell
  • must be able to replicate independently
  • have several restriction enzyme sites to allow insertion of DNA fragments using ligase
  • carry selectable gene marker to distinguish host cells that have taken them up from those that have not
34
Q

plasmid

A
  • extrachromosomal double-stranded DNA molecule

- replicates independently from chromosomes within bacterial cells

35
Q

plasmids used in DNA cloning

A
  • genetically modified bacterial plasmids - first vectors developed
  • engineered to contain: a number of convenient restriction sites and a marker gene to select for presence in host cell
36
Q

DNA ligase

A
  • DNA fragments will seal phosphodiester backbone

- joins restriction fragments covalently to produce intact DNA molecules

37
Q

transformation

A
  • plasmids are introduced into bacteria via transformation

- two main techniques: using calcium ions and brief heat shock to pulse DNA into cells and electroporation

38
Q

electroporation

A

a brief but high intensity pulse of electricity to move DNA into bacterial cells

Genetics (11 decks)

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