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Molecules to Cells - Genetics and Virology > Lecture 23. DNA III > Flashcards

Lecture 23. DNA III Flashcards

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1
Q

What can DNA be chemically synthesised by ?

A

Oligonucleotides

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2
Q

What is the structure of oligonucleotides ?

A

They are short (21-29 bases) single stranded, of defined base sequence

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3
Q

What are synthetic oligonucleotide primers key to the development of ?

A

PCR and DNA sequencing

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4
Q

What an a pair of oligos be used for ?

A

To prime the amplification of a target DNA sequence in a cyclical manner

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5
Q

How is a target sequence of DNA amplified ?

A
  1. DNA strands separated by heating to 98 degrees celsius
  2. Temperature is reduced to allow oligo primers to anneal (recombine DNA)
  3. Reduce temperature, add dNTPs and DNA polymerase
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6
Q

What does a PCR use and from where ?

A

A heat stable DNA polymerase from thermophilic bacterium

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7
Q

What is reverse transcriptase ?

A

Makes complementary DNA from RNA template

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8
Q

What is a major application of PCR ?

A

Genotyping of alleles based on allele specific length difference

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9
Q

What is multiplex genotyping ?

A

Using fluorescently labelled primers designed to amplify alleles of different sizes. These can be separated on the same gel

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10
Q

What is the advantage of multiplex genotyping ?

A

Shows alleles at many different loci to be investigated simultaneously

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11
Q

What are the major application of multiplex genotyping ?

A
  1. Forensic science
  2. Paternity testing
  3. Population genetics
  4. Evolutionary genetics
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12
Q

Who developed the DNA sequencing “sequence by synthesis” method ?

A

Fred Sanger

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13
Q

In sequencing by synthesis, what defines the start point ?

A

The site to which the primer anneals

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14
Q

What extends the primer in the sequencing by synthesis method?

A

Adding dNTPs and DNA polymerase

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15
Q

What and how separates the DNA strands in sequencing by synthesis method ?

A

DNA strands are separated by size using gel electrophoresis

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16
Q

What is the gel used in gel electrophoresis in the sequence by synthesis method ?

A

Polyacrylamide gel

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17
Q

In the sequence by synthesis method, what is used to reveal the newly synthesized DNA strands ?

A

Autoradiography

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18
Q

What was key to Sanger sequencing ?

A

The development of di-deoxynucleotides

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19
Q

What is the function of di-deoxynucleotides in Sanger sequencing ?

A

Terminate chain synthesis and gels to resolve DNA fragments of similar size

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20
Q

What prevents/terminates the elongation of the chain in sequence by synthesis method?

A

The absence of the 3’-OH group in di-deoxynucleotide

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21
Q

What makes sequencing more efficient in sequencing by synthesis method ?

A

Fluorescently labelled di-deoxynucleotides

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22
Q

What causes bands to fluoresce in sequence by synthesis method ?

A

Lasers

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23
Q

What is each di-deoxynucleotide tagged with in sequence by synthesis method sequencing ?

A

A unique colored fluorophore

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24
Q

What is next generation sequencing ?

A

Very fast, parallel short reads of millions of 35-400 base pair sequences

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25
Q

What is shot gun sequencing ?

A

Sequence of millions of small random fragments assembled by computers

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26
Q

Where are genes located ?

A

In the nucleus

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27
Q

Where are proteins/polypeptides synthesised ?

A

In the cytoplasm

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28
Q

What is each protein specified by ?

A

One gene

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29
Q

Where is information exchanged between in eukaryotes ?

A

The nucleus and cytoplasm

30
Q

What evidence is there to suggest that there is an intermediate in which the DNA in the nucleus specifies the production of proteins in the cytoplasm ?

A
  1. DNA is located in the nucleus but proteins are made in the cytoplasm
  2. RNA is synthesised in the nucleus
  3. RNA migrates to the cytoplasm where proteins are synthesised
  4. In viral infections, RNA synthesis precedes new viral particle formation
31
Q

What is the structure of RNA

A
  1. A polymer with a ribose-phosphate backbone
  2. Ribonucleic acid
  3. Bases (uracil instead of thymine)
32
Q

Is RNA usually single or double stranded ?

A

RNA is usually single stranded but could form double helices, but the complementary strand is not usually present in cells

33
Q

What is the difference between RNA and DNA

A
  1. Uracil in RNA, thymine in DNA
  2. Ribose in RNA, 2-deoxyribose in DNA
  3. RNA is single stranded, DNA double stranded
34
Q

What are the three major types of RNA

A
  1. Messenger RNA
  2. Ribosomal RNA
  3. Transfer RNA
35
Q

What is messenger RNA ?

A

RNA transcribed from protein coding genes

36
Q

What is the abundance of mRNA in a cell ?

A

Low abundance, less than 10%

37
Q

Does mRNA have a short half life ?

A

Yes

38
Q

What is rRNA ?

A

A structural and functional component of ribosomes

39
Q

Is rRNA abundant in cells ?

A

The most abundant form of RNA, greater than 90% of total RNA

40
Q

Is rRNA stable or unstable ?

A

Stable

41
Q

What are some characteristics of tRNA ?

A
  1. Very small (less than 100 bases long)
  2. Plays a key role in translation
  3. Very abundant and stable
42
Q

What are the different types of RNA polymerase in prokaryotes ?

A

A single RNA polymerase

43
Q

What are the different types of RNA polymerases in eukaryotes and their functions ?

A
  1. RNAP 1 - Makes rRNA
  2. RNAP II - Makes mRNA
  3. RNAP III - Makes tRNA
44
Q

How does RNA polymerase know where to start making mRNA and to stop ?

A

There are signals in the DNA sequence

45
Q

What is the transcription initiation site called ?

A

+1

46
Q

What direction does transcription occur ?

A

Heading downstream/ from left to right

47
Q

What is another name for the template strand ?

A

The coding strand

48
Q

What are promoters ?

A

DNA sequences that guide RNA polymerase to the beginning of a gene to start transcription (initiation site)

49
Q

What are terminators ?

A

DNA sequences that specify the termination of RNA synthesis and release of RNA polymerase from the DNA

50
Q

What are the three steps of RNA synthesis ?

A
  1. Initiation of transcription
  2. Elongation
  3. Termination
51
Q

What do prokaryotic promoters contain ?

A

2 highly conserved regions

52
Q

Does RNA polymerase bind to similar regions in the promoters if many different genes ?

A

Yes

53
Q

What are the roles of RNA polymerase ?

A
  1. Scans the DNA to identify a promoter
  2. Initiates transcription of a mRNA
  3. Elongates the mRNA chain
  4. Terminates transcription
  5. Interacts with regulatory proteins (activators and repressors)
54
Q

What and where do promoters usually consist of ?

A

Consist of a 40 base pair region located upstream of the transcription start site

55
Q

What are the promoters 2 conserved sequence elements and the consensus ?

A
  1. The “-35 region”, with consensus TTGACA

2. The “-10 region”, with consensus TATAAT

56
Q

What are some characteristics of the core RNAP ?

A

Sequence independent, non-specific transcription

57
Q

What is the core RNAP made up of ?

A

alpha, alpha, beta, beta prime

58
Q

What is the sigma subunit required for ?

A

Specific recognition of particles

59
Q

What is delta^70 ?

A

Vegetative (principle delta)

60
Q

What is delta^32 ?

A

Heat shock (for emergencies)

61
Q

What is delta^60 ?

A

Nitrogen starvation (for emergencies)

62
Q

How does RNA polymerase bind to DNA ?

A

Non-specifically

63
Q

What does RNA polymerase and the promoter form ?

A

A closed promoter complex

64
Q

How does an open promoter complex form ?

A

RNA polymerase unwinds about 12 base pairs from a closed promoter complex

65
Q

What does sigma bind ?

A

Elements centered around -35 and -10

66
Q

How does the termination of transcription in E. coli occur ?

A
  1. Simple

2. Complex

67
Q

In simple E. coli termination, is a protein factor required ?

A

No

68
Q

What happens in the simple termination of transcription in E. coli ?

A

A hairpin structure forms near the end of mRNA which causes RNA polymerase to dissociate

69
Q

What is the protein factor required in complex termination of transcription in E. coli ?

A

Rho

70
Q

What type of protein is Rho ?

A

A helicase protein

Molecules to Cells - Genetics and Virology (16 decks)

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