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BMS > Tools of Molecular Genetics > Flashcards

Tools of Molecular Genetics Flashcards

1
Q

Cut DNA molecules at specific sites

-Reckognizes short sequences of double stranded DNA up to 4-8 bps

A

Restriction Nucleases

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2
Q

Recognize palindromic (read the same 5’ to 3’) sequences

A

Restriction Nucleases

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3
Q

Once restriction enzymes recognize the pallindromic sequences, they cut in which two ways?

A
  1. ) Blunt ends

2. ) Sticky ends

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4
Q

A cut at the center of the recognition sequence results in

A

Blunt ends

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5
Q

The sticky end cuts are very useful when we are trying to

A

Clone DNA

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6
Q

RNA gel electrophoresis is similar to DNA gel electrophoresis with what exception?

A

RNA forms extensive secondary structure

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7
Q

The extensive secondary structure that RNA forms prevents it from migrating strictly according to its size. How do we get around this?

A

Add denaturants such as formamide

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8
Q

What do we use to visualize the electrophoresis gels of DNA and RNA?

A

Ethidium bromide staining or radioisotope labeling

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9
Q

To visualize the DNA bands, the gel is soaked in a dye (such as ethidium bromide) that binds to DNA and fluoresces brightly under

A

UV light

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10
Q

An alternative method for visualizing DNA is

-The exposure of x-ray film to a radioactive sample

A

Autoradiography

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11
Q

DNA can be labeled with radioactive isotope

-Will expose autoradiographic film

A

P32

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12
Q

Allows us to compare and analyze DNA and RNA molecules of identical or related sequences

A

Hybridization

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13
Q

Advantageous for detecting specific species of DNA or RNA in a mixture and estimating the quantities of each

A

Hybridization

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14
Q

The single stranded DNA or RNA used to detect the unknown DNA is called a

A

Probe

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15
Q

How can we denature the double stranded DNA or folded RNA so that we can hybridize with our probe?

A

Denature with either High temp or High pH

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16
Q

Once we add the probe, we can allow the DNA to reannel by

A

Lowering the temp or pH

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17
Q

We pick conditions to ensure that DNA or RNA anneals with our probe. How well these conditions allow our DNA or RNA to anneal together is called

A

Stringency of conditions

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18
Q

The higher the stringency, the

A

Lower the probability of hybridization

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19
Q

Increasing the temperature or amount of denaturing agent such as formamide raises the

-Lowers probability of hybridization

A

Stringency

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20
Q

If we want only a very specific fragment to anneal with a probe, we can adjust conditions accordingly. For example, we could

A

Raise temp or concentration of formamide

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21
Q

If we want to anneal heteroduplexes with mismatches, we can use

A

Lower temp or lower conc. of formaldehyde

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22
Q

Another way to manipulate stringency is through the length of the

A

Probe

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23
Q

Form stable heteroduplexes with target sequences that are similar but not identical to the probe

A

Longer nucleic acid sequences (more than 100 nucleotides long)

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24
Q

Allows cross-species analyses and identification of distantly related members of a gene family

A

Reduced stringency

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25
Q

Less tolerant of sequence mismatches than long probes

A

Short probes

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26
Q

Makes it so that it is possible to select for perfectly matched duplexes only

A

Short probes (High stringency)

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27
Q

Long probes can form stable hybrids even in the presence of

A

Mismatches

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28
Q

Allow you to distinguish between allelic sequences that differ by just a single nucleotide

A

Short probes (High stringency)

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29
Q

An inherited difference in the pattern of restriction enzyme digetion

A

Restriction Fragment Length Polymorphism (RFLP)

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30
Q

In order to detect RFLP we need to use

A

Restriction fragments

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31
Q

The target molecule of interest in southern blotting is

A

DNA

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32
Q

Used to detect specific DNA fragments

A

Southern Blotting

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33
Q

Useful in investigating the number of copies of a gene or whether there are large deletions, insertions, or rearrangments

-Can also be used to detect a point mutation

A

Southern Blotting

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34
Q

Describe a southern Blot

A
  1. ) Unlabeled DNA cut w/ restriction enzyme
  2. ) DNA fragments separated by agarose gel electrophoresis
  3. ) Fragments blotted onto nitrocellulose paper
  4. ) Labeled DNA probe hybridized to separated DNA
  5. ) Sheet is washed so that only hybridized DNA fragments remain
  6. ) Labeled hybridized fragments visualized by autordiography
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35
Q

An adaptation of southern blotting to detect specific sequences in RNA

A

Northern Blotting

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36
Q

The radioactive probe for Northern Blotting is usually a

A

Single stranded cDNA molecule

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37
Q

Allow us to monitor gene expression levels

A

Northern Blots

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38
Q

More efficient way than a Northern Blot to monitor gene expression levels

A

DNA microarrays

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39
Q

Used to monitor the expression of many thousands of genes simultaneously

A

DNA microarrays

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40
Q

How do DNA microarrays work?

A

Reverse transcribed mRNA is turned into cDNA labeled with fluorochrome and hybridized to a microarray. The colored spots on the microarray will show which gene is expressed at a higher level

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41
Q

Designed to match the nucleotide sequence flanking the substitution

A

Allele-specific Oligonucleotides (ASOs)

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42
Q

Patient DNA is hybridized to a panel of mutation specific probes

A

Allele specific Blotting

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43
Q

Allele specific blotting is used to detect things like

A

Sickle cell mutations and as a strategy for screening for thalassemias

44
Q

Simple and rapid technique to amplify the sequence of a target DNA up to 10^9 fold in just a few hours

A

Polymerase Chain Reaction (PCR)

45
Q

A valuable tool in medicine because it allows for the ampliphication of even trace amounts of DNA

A

PCR

46
Q

PCR is a repetition of many cycles consisting of three steps. What are the three steps?

A

Step 1.) Heat to separate DNA strands
Step 2.) Cool and add primers (annealing)
Step 3.) Primers are incubated w/ DNA polymerase and the four deoxynucleotides, and complementary strands are synthesized

47
Q

In order to perform PCR we need to design two

A

Primers (one for each strand)

48
Q

Direct the amplification of the desired piece of DNA

A

PCR Primers

49
Q

PCR depends on usage of a heat stable

A

Polymerase (taq polymerase)

50
Q

What amplification does each cycle of the PCR have?

A

Each cycle doubles the number of strands from the previous cycle

51
Q

Uses repeat rounds of strand separation, hybridization, and synthesis to amplify DNA

A

PCR

52
Q

Dependent on perfect base-pairing of the 3’ end of nucleotide primers

A

Allele specific PCR

53
Q

Allows us to selectively amplify a mutant allele cause by a mismatch

-because the designed primer will bind to the mismatch much better than taq polymerase

A

Allele specific PCR

54
Q

mRNA is isolated from a tissue, and then a cDNA is synthesized using a reverse transcriptase. The original RNA template is removed by RNAse H and cDNA is amplified. PCR then occurs

A

Reverse Transcriptase PCR (RT-PCR)

55
Q

Removes the RNA tamplate in RT-PCR

A

RNAse H

56
Q

In this technique, a target DNA strand is replicated in vitro using a primer, DNA polymerase, and a mixture of dNTPs for adenine, guanine, thymine, and cytosine in four different reaction tubes. Each reaction tube also contains a small amount of one of the four radiolabeled nucleotide analogs

A

DNA Sanger sequencing

57
Q

How does Sanger sequencing work?

A

Randomly, the radioactive nucleotide analogs will be inserted into the newly synthesized strand and synthesis will stop. This allows us to see which nucleotide occupies which position in the target DNA

58
Q

The nucleotide analogues lack the

A

3’ OH needed for synthesis to continue

59
Q

When the Sanger sequencing reaction is complete, each tube contains a mixture of radioactively labeled

A

DNA fragments of different length

60
Q

The bottom of the gel for Sanger sequencing represents the

A

5’ End of the newly synthesized DNA

61
Q

The top of the gel for Sanger sequencing represents the

A

3’ end of the newly synthesized strand

62
Q

The strand synthesized by Sanger sequencing will be complementary to our

A

Target strand

63
Q

A single reaction tube will contain the target DNA, a primer, all four dNTPs and all four analogues

A

Automated DNA sequencing

64
Q

In automated DNA sequencing, the PCR reaction will generate the DNA fragments that are separated by capillary electrophoresis. The fluoresence emission of each peak is monitored by a laser detector and recorded on a chromatogram where the 5’ end of the sequence is on the

A

Left

65
Q

Recombinant DNA can be copied inside of

A

Bacterial Cells

66
Q

Some bacteria can efficiently take up foreign DNA from their surroundings, a phenomenon called

A

Transformation

67
Q

Plasmids that are taken up by the host bacteria are maintained as a piece of DNA independent of the bacterial chromosomes. Therefore, they are useful tools because their replication is independent of the

A

Host Cell

68
Q

Specialized plasmid vectors are used to

A

Clone DNA

69
Q

How do we clone DNA using plasmids?

A

Cut open circular plasmid and insert the DNA fragment to be cloned. When the resulting recombinant DNA replicates, our fragment will be cloned

70
Q

Cleaves the circular double-stranded plasmid (vector) DNA for insertion of the DNA fragment we want to clone

A

Restriction Nuclease

71
Q

The DNA fragment we want to clone is covalently linked to the vector by

A

DNA ligase

72
Q

The DNA fragment we wish to clone must be cut with the same

A

Restriction enzyme

73
Q

The recombinant plasmid is made inside of a test tube. When we introduce the recombinant plasmid DNA into a bacterial cell, what happens?

A

The plasmid will be replicated millions of times

74
Q

We can use DNA cloning by plasmid vectors to compile a

A

DNA library

75
Q

A collection of cloned fragments of an organism

  • Two types
    1. ) Genomic
    2. ) cDNA
A

DNA libraries

76
Q

Libraries of human genomic DNA fragments can be constructed using

A

Restriction nuclease and ligase

77
Q

A bacterial colony carrying a particular DNA clone can be identified by

A

Hybridization

78
Q

When compiling a genomic library, once we have transferred our bacterial colonies to a sheet, we can lyse the bacteria and denature the DNA with

A

Alkali

79
Q

After we have denatured the DNA, we then add radioactively labeled DNA probes specific for the plasmid of interest. Then we expose the paper to

A

Photographic film

80
Q

Living cells containing the plasmid of interest can then be isolated from the colony disk and

A

Grown in large quantities

81
Q

Represent the mRNA produced by a particular tissue

A

cDNA libraries

c = complimentary

82
Q

cDNA is double stranded DNA synthesized from RNA by

A

Reverse transcriptase and DNA polymerase

83
Q

Genomic DNA clones and cDNA clones derived from the same region of DNA are

A

Different

84
Q

Exons, introns, and non-transcribed DNA are included in the DNA clones of the

A

Genomic Library

85
Q

The intron sequences are removed and a continuous coding region is present in each clone of the

A

cDNA library

86
Q

Allows you to see which genes are expressed more frequently

A

cDNA Library

87
Q

Genes will be represented equally regardless of their levels of expression in the

A

Genomic Library

88
Q

Can be used to splice together a set of DNA fragments derived from different sources (making Chimeras)

A

Serial DNA cloning

89
Q

Why do we need to make chimeric proteins?

A

Adding a fluorescent protein to a protein of interest allows us to visualize where the protein is located in the cell

90
Q

In serial DNA cloning, after each DNA insertion step, the recombinant DNA is

A

Cloned (purifies sample)

91
Q

The purified cloned DNA is then cut with a restriction nuclease, and

A

Another DNA fragment is added

92
Q

Can be produced from a protein-coding sequence cloned into and expression vector and introduced into cells

A

Large amounts of protein

93
Q

To use DNA cloning to make a protein, we first must make a vector plasmid containing a

A

Highly active promoter for the protein of interest

94
Q

Make it possible to move experimentally from gene to protein and from protein to gene

A

Recombinant DNA techniques

95
Q

If we want to make DNA from a protein, we must determine the amino acid sequence of a purified peptide fragment, then we want to

A

Search the DNA database for the gene sequence

96
Q

Used to determine the pattern of a gene’s expression

A

Reporter Genes

97
Q

Let’s say we want to find out which cell types express protein “X” but it is difficult to detect the protein “X” directly, what can we do?

A

Replace the coding sequence for protein “X” with a reporter gene that expresses a protein like GFP which can be easily monitored

98
Q

The reporter protein will only be expressed in places where

A

The target protein is expressed

99
Q

In order to tell which regulatory regions control expression in particular cell types, we

A

Turn off all but one sequence at a time and see where the protein is expressed

100
Q

We can add epitopes as

A

Reporters

101
Q

In order to create an epitope tagged protein, we fuse the gene for the epitope tag to the gene for the

A

Target Protein

102
Q

We can use DNA cloning and recombinant DNA technologies to genetically modify animals by

A

Gene replacement, gene knockout, and gene addition

103
Q

When the normal gene is entirely replaced by a mutant copy of the gene

-provides information on the activity of the mutant gene without interference from the normal gene

A

Gene replacement

104
Q

The normal gene can be completely inactivated by making large deletions in it. This process is called

-Widely used to obtain information on the function of the normal gene

A

Gene knockout

105
Q

A mutant gene can be added to the normal genome. This provides information when the introduced mutant overrides the function of the normal gene

A

Gene addition

BMS (62 decks)

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