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Molecular Biology techniques for cell biology Flashcards

module 2 (63 cards)

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1
Q

What is polymerase chain reaction (PCR) widely used for?

A

to clone genes

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2
Q

how does polymerase chain reaction work?

A
  1. must know the sequence of DNA what to amplify
  2. Gene specific primers complementary to gene of intreats amplify sequence
  3. amplified DNA can be cloned
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3
Q

what are restriction fragments used for?

A

used to experimentally for recombinant DNA technology

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4
Q

what are the steps for restriction fragments?

A
  1. DNA from 2 sources treated with restriction enzyme to generate fragments with sticky ends
  2. fragments mixed together under conditions that favour base pairing btwn sticky ends
  3. cut molecules are sealed together by DNA ligase
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5
Q

what is the basis of restriction enzyme job?

A

to cut and recombine DNA

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6
Q

what does restriction endonuclease do?

A

cut DNA molecules at specific internal sites

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7
Q

what is the recognition sequence called in restriction endonucleases?

A

restriction site

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8
Q

what is the formation of the restriction site?

A

palindrome (antiparallel)

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9
Q

when restriction enzymes cut at the same point, what does it make?

A

blunt ends

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10
Q

how do restriction enzymes make sticky cuts?

A

cut at staggered sites

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11
Q

what does recombinant DNA technology allow?

A

allows scientists to create recombined DNA molecules with DNA from different sources

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12
Q

how are proteins studied through recombinant DNA technology?

A

can be studied and manipulated indirectly by manipulating DNA sequences that encode them

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13
Q

what is DNA cloning?

A

generation of many copies of a specific DNA sequence

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14
Q

what is DNA transformation?

A

process of introducing DNA into the cell

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15
Q

what does gel electrophoresis allows?

A

allows DNA to be separated by size

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16
Q

how does gel electrophoresis work?

A

DNA molecules are negatively charged therefore will migrate towards the anode (positively charged electrode)

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17
Q

what is the DNA detected with in gel electrophoresis?

A

ethidium bromide (stain), can see under UV light

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18
Q

what does DNA sequencing make possible?

A

to analyze the entire genome of an organism

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19
Q

what does genome wide sequence analysis do?

A

gives variety of types of information

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20
Q

what is pyro sequencing?

A

sequencing through light

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21
Q

how does pyro sequencing work?

A

four labeled nucleotides and firefly enzyme luciferase
incorporation of correct nucleotide on strand releases pyro phosphate which releases light when interacts with luciderase

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22
Q

what is illumination sequencers?

A

sequencing through fluorescents

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23
Q

what does specializes dNTPs do?

A

tag a unique fluorescent signal for each base

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24
Q

what is a reverse terminator do?

A

blocks the addition of new bases

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25
what happens when a base is added with Illumina sequencers?
it can be identified in real time by florescence emissions
26
when a base is identified in illumina sequencers, what is the next step?
the terminator is removed, another base added and identified
27
what is third generation sequencing?
nano pre sequencing
28
what is nano pore sequencing?
produces long reads but doesn't have a DNA synthesis step
29
what are the steps to nano pore sequencing?
1. protein pores embedded in artificial membrane 2. single stranded DNA goes through, disrupts electrical field 3. each base disrupts the electrical field differently (allows base identification)
30
how are bases identified through nano pore sequencing?
when base disrupts electrical field, it has own electrical field signature
31
what does bioinformatics do?
helps decipher genomes
32
how does Bioinformatics work?
merges computer science with bio to organize sequencing and other data
33
what is an exome?
part of genome that contains exons (encoded proteins)
34
what is transcriptone?
the entire set if RNA molecules produced by a genome
35
what is a proteome?
all the proteins produced by an organism. it'a more complex than the genome
36
what is the percent of the genome all humans share?
99.7%
37
what is the percent of the genome that varies person to person?
0.03%
38
what does inherited diseases result from?
mutations in single loci (genes)
39
what are multi genetic diseases?
many different genes affect the likelihood that the disease will arise
40
how is RNA analyzed?
RT-PCR RNA-SEQ
41
what does RT-PCR help you know?
necessary to know when and where RNAs are expressed
42
how does RT-PCR work?
cDNA is made from RNA through reverse transcription cDNA amplified by PCR using primers
43
what is RT-PCR for?
indirectly determine whether an mRNA present in a sample
44
what is another name for RNA-SEQ (RNA sequencing)
also called whole transcriptome shotgun sequencing
45
where is the data assembled from RNA-SEQ?
bioinformatics tool
46
what is transcrtptomics?
study of the quantities and functions of all the RNAs present in a particular gene
47
how are proteins analyzed?
SDS polyacrylamide gel electrophoresis western blotting with antibodies dissecting protein function via mutations
48
what is SDS polyacrylamide?
used to separate proteins with heat and anionic detergent SDS
49
what are the steps of SDS polyacrylimide gel electrophoresis?
1. proteins are solubilized 2. loaded into wells of gel 3. electrical potential is applied (anode at bottom) 4. rate of movement related to their size (small travel fast) 5. the gel is stained with dye to visualize proteins
50
how are specific proteins detected?
through western blotting with antibodies
51
what are the steps of western blotting with antibodies?
1. protein mixture is solubilized and separated by SDS PAGE 2. then blotted into membrane using electrical current 3. blot incubated with primary anti-bodies and then secondary antibodies 4. secondary attached to enzyme can be detected by coloured precipitate or through chemiluminescence
52
what is dissecting protein function via mutations?
pieces of DNA encoding a protein can be deleted, leaving DNA that encodes only part of protein
53
what does examining the protein fragment do in dissecting protein function?
helps determine which parts of the protein are important for the function
54
what is site-directed mutagenesis?
single base can be mutated so the resulting protein is altered
55
how is gene function analyzed?
genetic engineering crispr genome editing
56
what is genetic engineering?
genetic engineering of proteins to in living organisms requires engineering the organism
57
what is a transgenetic organism contain?
foreign pieces of DNA that can be passed to subsequent generations
58
what is the process to create organisms called transgenesis?
1. direct injection of bombardment 2. infection (in plant bio technology)
59
what is crispr genome editing?
technologies to alter the genomes of cells and organisms collectively involve genome editing
60
how does CRISPR work?
sequences are used to target complexes to certain genomic sites were the genomic DNA is directly altered
61
what happens to double stranded breaks in CRISPR genome editing?
they are induced then repaired
62
what is the result of CRIPR genome editing?
the result id removal of the DNA or replacement of normal DNA with another sequence
63
what is the molecular biology applications of CRISPR genome editing?
guid RNA acts along with crRNA and CRISPR associated protein (Cas 9) to introduce double stranded breaks on foreign DNA

Cell Biology (3 decks)

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