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GEN > GEN3 - Defining the Genome II (DNA) > Flashcards

GEN3 - Defining the Genome II (DNA) Flashcards

1
Q

What is the transforming principle?

A

Idea of how one strain can become pathogenic through picking up something from another strain.

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2
Q

What were the four experiments trialled that indicated the transforming principle? Give a conclusion for each.

A

Live rough strain cells - no mice death - R strain not pathogenic.
Live smooth strain cells - mice death - S strain pathogenic.
Heat killed S strain cells - no mice death - heat used to kill mucus coating therefore mucus not pathogenic as mice did not die.
Heat killed S cells and living R cells - mice death - Live S cells found indicating that heat killed S cell had caused live R cell to transform into S cells.

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3
Q

What was the main further experiment/conclusion that came about as a result of the transforming principle discovered by Griffiths?

A

Individual components of DNA, protein, RNA and lipids of the S cell were trialled to see effect on mice. Death occurred only in case of DNA lysate, indicating that is responsible for transforming principle.

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4
Q

Following identification of DNA being the key molecule for genetic information- what was then produced? Give examples of these.

A

Various methods for DNA analysis e.g. Sanger sequencing, recombinant DNA cloning, NGS, restriction enzymes etc.

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5
Q

What is the purpose of Sanger sequencing?

A

Sequencing of nucleotides within a DNA sequence fragment.

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6
Q

What reagents are required for Sanger sequencing?

A 
Purified target DNA sample. 
DNA polymerase.
Oligonucleotide primer.
dNTPs.
ddNTPs
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7
Q

What is the difference between dNTPs and ddNTPs? What is the effect of this?

A

ddNTPs lack the 3’-OH group that dNTPs contain. This prevents chain elongation meaning that every time a ddNTP is incorporated into a chain - chain termination occurs.

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8
Q

If different peaks are produced in a Sanger sequencing spectrum, what might this indicate?

A

Copy number - higher peaks have a larger number of copies.

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9
Q

Give an overview of the process of Sanger sequencing.

A

Primer anneals to DNA fragment. DNA polymerase elongates chain using dNTPs. If ddNTP is used, then chain is terminated. Every possible combination of chain length is produced. Sequencing machines are then used to determine the chain sequence. Measures the fluorescence as fluorescent fluorophores are added to each of the ddNTPs - different colours for each type.

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10
Q

What is the maximum size of the DNA fragment that can be sequenced using Sanger sequencing - in one go?

A

Up to 1000 nucleotides

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11
Q

What are the differences between exonucleases and endonuclease?

A

Exonuclease - cuts DNA at the ends I.e. either from the 3’ or 5’ ends.
Endonuclease - cuts DNA within a sequence allowing for formation of two (or more) fragments.

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12
Q

What are restriction enzymes and what do they do?

A

Endonuclease enzymes obtained from different bacteria. Recognised (palindromic) sequences to allow for cleaving double stranded DNA, either producing blunt ends or sticky overhangs.

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13
Q

What is the purpose of bacteria containing restriction enzymes?

A

Form part of bacterial immune response. Cuts DNA of viral infecting cells. Prevents replication of viral DNA and ability to attack host cells.

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14
Q

Discuss the sequence length of the recognition site of a restriction enzyme, and how often this appears in the human genome.

A

Restriction sites are approx 4-8 base pairs long. How long it is determines its abundance within the genome.

E.g. of sequence is 5 nucleotides, and taking into account there are 4 possible nucleotides, the exact sequence would once every 4^5 base pairs.

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15
Q

Give three examples of restriction enzymes, what bacteria they are obtained from, and their resulting effects.

A

HindIII - from haemophilis influenza, cuts at palindromic AAGCTT, produces 5’ sticky ends
PstI - from providencia stuartii, cuts at palindromic CTGCAG, produces 3’ sticky ends
EcoRV - from E. coli, cuts at GATATC, produces blunt ends

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16
Q

Discuss link between DNA methylation and use of restriction enzymes.

A

Some restriction enzymes are affected by DNA methylation - their activity is either inhibited or completely blocked as a result of methylation.

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17
Q

Give one use of restriction enzymes.

A

Recombinant DNA cloning.

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18
Q

Give brief overview of recombinant DNA cloning procedure.

A

Restriction enzyme used to cut specific DNA sequence of interest - produces sticking overhangs. Same restriction enzyme used to cut plasmid DNA - produces complementary sticky overhangs. DNA ligase used to seal together the complementary sticky overhangs.

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19
Q

Give one use of recombinant DNA cloning.

A

Used for production of proteins large scale - e.g. diabetic insulin production.

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20
Q

How can the recombinant plasmids be selected for during recombinant DNA cloning?

A

Plasmids either need to be screened using PCR or if antibacterial resistance gene incorporated at same time as gene of interest - allows for detection by placing bacteria within antibiotic culture.

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21
Q

Discuss the use of recombinant DNA cloning in genome studies.

A

Genome undergoes fragmentation. Fragments are added into plasmids using recombinant DNA technology. Bacteria containing recombinant plasmids are left to reproduce. Large amounts of DNA are produced which can undergo Sanger sequencing. Genomic library can be created.

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22
Q

What is southern blotting used for?

A

Detection of specific DNA sequences..

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23
Q

Discuss use of Southern, Western and Northern blotting.

A

Southern - DNA
Western - protein
Northern - RNA

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24
Q

What are some potential uses of Southern blotting?

A

Biomedical research, clinical diagnoses and testing, paternity testing, forensic analyses.

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25
Q

Give brief overview of process of southern blotting.

A

DNA fragmented. Electrophoresis used to sort fragments by size. DNA transferred to membrane. Probes used to visualise the DNA fragments as the probes are complementary to the DNA and are attached to fluorescent dye/probe.

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26
Q

How can southern blotting be used for clinical diagnosis? Give one example.

A

Can be used for diagnosis of sickle cell disease. MstII restriction enzyme cuts DNA twice in sickle cell allele but three times in normal allele. Different numbers of DNA a fragments will appear on southern blot depending on whether the individual is unaffected, affected or a carrier.

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27
Q

What type of vectors can be used recombinant DNA technology?

A

Can use both plasmids and bacteriophages.
Plasmids - readily available, no risk, suitable for smaller fragments only.
Bacteriophages - much larger so more useful for larger DNA fragments.

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28
Q

Give the reagents required for PCR.

A

DNA template fragment - purified.
Forward and reverse oligonucleotide primers - complementary to DNA sequence of interest.
Heat stable DNA polymerase enzyme.
dNTPs

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29
Q

What are the three stages in one cycle of PCR? Give approx temperatures for each stage.

A

Denaturing - unwinding of DNA double helix by breaking H bonds between the strands. Approx 95 degrees.

Annealing - formation of H bonds between primers and single stranded DNA. Approx 55 degrees.

Extending - heat stable DNA polymerase uses dNTPs to elongate the fragment from the primer. Approx 72 degrees.

30
Q

Why is a heat stable DNA polymerase required?

A

Higher temperatures are required within PCR to ensure H bonds are overcome and for faster reactions - if non heat resistant DNA polymerase was used, it would become denatured.

31
Q

What is the amplification factor of PCR?

A

2 to the power of n where n is the total number of cycles.

32
Q

What is the main use of PCR? Give examples of when this use is required.

A

Amplification of a DNA fragment.

Used for basic research, clinical diagnoses and forensic analyses.

33
Q

Give one advantage of PCR over southern/northern blotting or molecular cloning.

A

Faster detection of DNA sequences.

34
Q

What is the key difference between qPCR and PCR?

A

qPCR records the number of cycles required for a specific set threshold of amplification to be reached, this is measured following every cycle.

PCR sets a specific number of cycles and measures the amplification at the end,

35
Q

What is the main advantage of NGS over Sanger sequencing?

A

Much faster.

36
Q

Give a brief overview of NGS process.

A

DNA fragmented into small fragments, adaptors are added to the fragments. These are then attached to the flow cell via complementary adaptors. By formation of a bridge, the fragments attach to primers. PCR is used to extend these fragments, via bridge amplification. One strand is then washed off, leaving multiple clusters, each with a single fragment (repeated many times). Sequencing can then be performed using fluorescently labelled nucleotides. Following the addition of each nucleotide, the colour can be observed.

37
Q

Give four reasons that contribute to the speed of NGS.

A

Solid support used for DNA cluster amplification - provides larger surface area.
Optimising reagents - ensures that fragment is elongated one nucleotide at a time.
Chain terminating groups and fluroescently labelled tags - allows easy detection,
Laser scanning - continuous laser scanning.

38
Q

Give four applications of NGS.

A

Whole genome sequencing.
Targeted sequence I.e. within specific genomic region.
Transcript one sequencing I.e. RNA seq which is quicker and more effective than northern blotting.
Chromatin immunoprecipitation sequencing - DNA protein interactions.

39
Q

Discuss the two main components of the genome.

A

Genes and gene related DNA.

Highly repeated DNA.

40
Q

What are the two types of highly repeated DNA a and the subcategories within them?

A

Dispersed transposons - LINEs, SINEs, LTR transposons, DNA transposons.
Tandem satellites - macro, mini, micro.

41
Q

Is the majority of protein coding genes exons or introns?

A

Mostly introns. Exons only make up approx 1.5% of protein coding genes.

42
Q

What is alternative splicing and what are its effects?

A

Alternative removals of introns I.e. may remove some exons as well, part of intro may be retained. Causes mRNA and protein isoforms production. Different versions from the same gene.

43
Q

Give examples of types of alternative splicing.

A

Constitutive splicing - normal splicing with all introns removed and all exons kept.
Exon skipping - some exons may be missed I.e. will go from exon 1 to exon 3.
Intron retention - some introns are kept within the mRNA.
Mutually exclusive exons - some exons must be added to the mRNA with other exons.
Alternative 5’ splice site -

44
Q

Discuss the presence of non coding DNA in high vs low gene density areas.

A

High gene density - lots of genes with little non coding DNA in the intergenic regions.
Low gene density - few genes with lots of non coding DNA in intergenic regions

45
Q

What is a gene dessert?

A

Areas within the genome that are approx 1 Mb in size and contain no genes.

46
Q

Discuss gene density differences between human, drosophila, yeast and bacterial genomes.

A

Highest gene density - bacteria
Yeast
Drosophila
Lowest gene density - humans

47
Q

Give five classes on non coding RNA.

A

Ribosomal, transfer, smaller nuclear, microbe and long non coding

48
Q

From approx what percentage of the genome is RNA transcribed from?

A

Approx 75%

49
Q

What are the four types of rRNA?

A

18S, 5.8S, 28S and 5S.

50
Q

Describe the structure of tRNA and its role in translation.

A

Small folders molecules of single stranded RNA. Holds the amino acid and brings to mRNA during translation. Synthesis of amino acid polypeptide chain.

51
Q

How are small nuclear or nucleolar RNA produced and give one common role for them?

A

Transcribed by RNA polymerase II or III. Common role - formation of splicesosome to ensure mature mRNA production from precursor mRNA.

52
Q

How is miRNA produced? What is the role of miRNA?

A

Transcribed by RNA polymerase II and III. Control gene expression by RNA interference e.g. physically blocking the access for the mRNA.

53
Q

Discuss the role of piRNA and give another type of RNA that it is similar to.

A

Piwi interacting RNA. Similar to miRNA. Uses process of RNA interference in germ line cells to silence transposons.

54
Q

What is lncRNA ? What is a common example of it?

A

Non coding RNA greater than 200 nucleotides. Commonly involved in mRNA expression. Common example is Xist.

55
Q

What are pseudo genes and what are the three types of pseudo genes? Briefly describe each type.

A

Non functional mutated genes formed as a result of genome evolution. Types include:
Non processed pseudogenes - duplicated functional gene which undergoes mutations and becomes not functional.
Processed pseudogenes - reverse transcription of spliced transcript. DNA produced is incorporated into genome.
Gene fragments - genomic rearrangements causing gene fragments to form which are non functional.

56
Q

Give a common product formed as a result of pseudogenes and one common example of this.

A

Pseudogenes commonly form gene families. E.g. beta globin gene family.

57
Q

What is receptive DNA? Give three common roles of it and the two main types that it is found as.

A

Large areas of non coding DNA which contains repeat regions and have various functions.
Functions - genetic variation, genetic regulation, genome folding.
Types - transposable elements, tandem repeats.

58
Q

Give a brief overview of what transposable elements are and give two types of it.

A

Transposable elements are also called transposons. Regions on DNA whose location changes within a genome.

Types - RNA and DNA transposons.

59
Q

What is transposition? Discuss the varying effects it can have in different cell types.

A

Transposition - movement of DNA region from one genomic location to another, commonly causing genomic modifications or chromosomal rearrangements. May occur in somatic cells or gametes.

Somatic cells - oncogenic effects.
Gametes - genetic diseases.

60
Q

Briefly describe the difference between DNA transposon retrotransposon.

A

DNA transposon - DNA removed from former transposon site and integrated into new target DNA region forming transposed mobile elements.
RNA transposon - DNA interacts with RNA polymerase to form RNA intermediate which undergoes reverse transcription into DNA which is then integrated into new target DNA region forming transposed mobile elements.

61
Q

How are DNA transposons integrated into new target genome regions?

A

DNA region has inverted repeat sequences flanking it. Allows integration into target DNA via transposase enzymes.

62
Q

What are the three types of retrotransposon?

A

LOng terminal repeats.
Long interspersed nuclear elements (LINEs).
Short interspersed nuclear elements (SINEs).

63
Q

What are the two main genes within LTR retro transposons, and their respective roles?

A

Gag - gene encoding cytoplasmic virus like particle

Pol - gene encoding reverse transcriptase enzyme

64
Q

What are LTR retro transposons commonly referred to? What is the key difference between retroviruses and LTR retrotransposon?

A

Commonly referred to as endogenous retrovirus (ERVs).
Key difference between retrovirus and LTR retrotransposon is that retrovirus contain env gene where LTR retrotransposon does not. Env controls ability to infect other cells.

65
Q

Describe why LINEs are considered autonomous whereas SINEs are non autonomous.

A

LINES are autonomous as they contain a reverse transcriptase gene within them. SINEs do not so would rely on another enzyme for their transposition.

66
Q

What ORFs are found in LINEs and what are there roles?

A

ORF1 - RNA brining protein important for transposition.

ORF2 - protein with endonuclease and reverse transcriptase activity.

67
Q

What are the three main types of tandem repeats?

A

Macro satellites.
Mini satellites.
Micro satellites,

68
Q

What is the key difference between transposons and tandem repeats?

A

Transposons are interspersed repeats whereas tandem repeats are repeated multiple times with one tandem repeat DNA a region.

69
Q

Which type of tandem repeat is used for DNA fingerprinting?

A

Mini satellites.

70
Q

Discuss the role of micro satellites in a specific disease.

A

Micro satellites can be located within protein coding genes eg Huntington protein. Where more than 35 repeats of CAG are found in this protein - Huntington’s disease occurs.

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