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Genomics > Lectures 6,7,8 COPY > Flashcards

Lectures 6,7,8 COPY Flashcards

1
Q

How large are DNA sequencing reads?

A
  • only 750 bp

- helpful for tracking down causative mutations for genetic diseases

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2
Q

What are the two approached to genome mapping?

A
  • Genetic mapping/linkage analysis - relies on the observation of inheritance patterns during genetic crosses
  • Physical mapping - using molecular biology techniques to directly identify features in the genome
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3
Q

Describe crossing over/recombination.

A
  • In prophase 1 of meiosis homologous chromosomes form a bivalent
  • Within the bivalent the chromosome arms or chromatids physically break and exchange segments of DNA
  • After recombination maternal chromosome 1 will have some genetic information from the paternal chromosome 1
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4
Q

What do semicolons indicate in dihybrid crosses?

A
  • a semi colon indicates that they are on different chromosomes
  • no semi colon indicates linked genes
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5
Q

What did thomas hunt morgan discover in reference to recombination?

A

genes are linked to each other and alleles can be uncoupled by recombination

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6
Q

What did Arthur Sturteuant discover?

A

1) Recombination is a random event - there is an equal chance that crossover occurs at any position between a pair of chromatids
2) Recombination frequency is therefore a measure of the distance between two genes

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7
Q

How does the distance between genes impact the frequency of recombination?

A
  • Further apart - high frequency of recombination

- Closer together - low frequency of recombination

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8
Q

What does it mean when two genes are linked?

A

they are located on the same chromosome

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9
Q

How is recombination measured?

A
  • percent recombination frequency = map units or centiMorgans (cM)
  • 1% = 1 map unit
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10
Q

What are some limitations to genetic mapping?

A
  • recombination hotspots -areas more likely to undergo crossover
  • chromatids can undergo multiple crossovers
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11
Q

How do you calculate recombination frequency?

A

the number of recombinant progeny / total progeny = % recombination frequency

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12
Q

Name 3 genetic markers.

A

1) Restriction Fragment Length Polymorphism (RFLPs)
2) Simple Sequence Length Polymorphisms (SSLPs)
3) Single Nucleotide Polymorphisms (SNPs)

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13
Q

What are RFLPs, SSLPs, and SNPs known as?

A

genetic markers

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14
Q

What is a restriction fragment length polymorphism (RFLP)?

A
  • when there is a restriction site only present on one allele - (Known as a polymorphic restriction site)
  • when different alleles are cut with restriction enzymes they create different numbers of fragments of DNA
  • this is detected through southern blotting
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15
Q

What is southern blotting?

A
  • Take genomic DNA sequence and cut it with restriction enzymes
  • Run them on a gel and get a smear
  • Copy gel to a nylon membrane
  • Incubate DNA probe with nylon membrane and it will bind to the DNA sequence complementary to it
  • Take autoradiograph to locate hybridizing bands (DNA sequence of interest)
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16
Q

What are SSLPs?

A
  • Simple sequence length polymorphisms
  • Two variants of an SSLP - two variants have different numbers of repeats of the same sequence
  • Microsatellites - repeats are 13bp or less
  • Mini satellites - repeats up to 25 bp in length
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17
Q

How are SSLPs identified?

A

can use PCR to identify how many base pairs/repeats there are on a DNA strand

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18
Q

What are SNPs?

A
  • single nucleotide polymorphisms
  • two different alleles have single base pair changes
  • one SNP for every 1000 bp - natural variation in the genome
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19
Q

How are SNPs identified?

A

through hybridization

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20
Q

What are three strategies for hybridization?

A 
A) Hybridization with an oligonucleotide with a terminal mismatch
-completely base-paired hybrid if no SNP
- hybrid with non-base paired tail - SNP
B) Oligonucleotide ligation assay
- no mismatch - ligation occurs
-mismatch - no ligation of DNA fragments
C) The ARMs test
-no mismatch PCR product is synthesized
- mismatch - no PCR product
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21
Q

What is the purpose of a DNA chip?

A

allows you to look at 300,000 SNPs from all across the genome

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22
Q

How are genetic markers helpful?

A
  • for forensic analysis
  • Allows you to search for if DNA from a crime scene and a suspect are the same without full sequencing
  • Could PCR certain fragments and cut the enzyme (RFLPs)
  • Could PCR for SSLPs
  • SNP analysis
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23
Q

What is a test cross?

A

an unknown x recessive,recessive -> allows genotypes to be deduced

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24
Q

What is FISH?

A
  • Fluorescent In-Situ-Hybridization
  • one way to physically map gene sequences onto genome of interest
  • Isolate chromosomes from cells, fix them onto a microsope slide, denature them, add probe (short DNA sequence that is complementary to the gene of interest)
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25
Q

What is restriction mapping?

A

cut DNA sequence of interest with different restriction enzymes - look at figure 3.28
-very useful for short fragments of DNA

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26
Q

What is optical mapping?

A
  • overcomes genome of interest unidentifiability problems
  • put chromosomal DNA on molten agarose containing restriction enzymes
  • agarose solidifies and DNA becomes stretched
    (Restriction enzymes cut in difference places, but DNA fragments stretch with the gel)
    -Fluorescence microscopy - DNA molecules with restriction sites visible
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27
Q

What is a clone library and how is one created?

A
  • A clone library is a collection of vectors carrying DNA fragments of several kilobases
  • Insert DNA fragments of different sizes into plasmids
  • Each colony contains multiple copies of one recombinant DNA molecule
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28
Q

How do you identify whether components of a clone library are closely linked or not?

A
  • PCR reaction to detect markers
  • If closely linked many clones will be positive for both markers
  • If far apart a clone would rarely test positive for both markers
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29
Q

What was Frederick Sanger’s first discovery?

A
  • determined the peptide sequence of insulin by degrading the sequence
  • tried to do this for DNA but degrading the sequence wouldnt allow you to solve the sequence of the molecule
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30
Q

What does traditional Sanger Sequencing involve?

A
  • incorporate a dideoxynucleotide
  • run four separate reactions with different DDNTPs
  • run the reactions on a gel to deduce their size
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31
Q

What does more modern sanger sequencing involve?

A
  • Label ddNTPs with fluorescent labels
  • Separate labelled fragments by size in a capillary and use a detector, which notes which fluorescent bands move past the detector and in which order
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32
Q

What is a ddNTP?

A

dideoxynucleotide - a nucleotide with a chemically altered base that has an -H in place of -OH on the 3’ end

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33
Q

What are some features of the polymerase used in Sanger sequencing?

A

1) High processivity - length of the polynucleotide that is synthesized before polymerase terminates - polymerase goes for a while before it falls off
2) No 5’-3’ exonuclease activity
3) No 3’-5’ exonuclease activity - could remove ddNTPs at the end

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34
Q

What are some examples of the primers used in Sanger sequencing?

A
  • Taq polymerase - processivity of 750bp
  • Forward, reverse and internal primers may be used
  • Universal primer - vector plasmid has primer site, located upstream of where DNA is inserted
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35
Q

How do you find where the genes are located in the genome?

A
  • to find which genes encode for protein start with mRNA and transcribe to DNA
  • RNA -> cDNA (coding DNA)
  • Use reverse transcriptase-polymerase that uses RNA as a template to make DNA
  • Can use cDNA sequence as a probe, and use a FISH experiment to find where this sequence is located in the genome
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36
Q

What is a radiation hybrid?

A
  • Radiation hybrid is a technique used to map the human genome
  • Expose the chromosome to be mapped to X-rays which allows it to become fragmented
  • Fragments are fused into a different species (possibly hamster)
  • Able to test hybrid cells for markers, if 2 markers are present in the hybrid it can be assumed they are close to each other because they likely crossed over together
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37
Q

What was the main challenge with sequencing the human genome?

A
  • the human genome is 3 billion base pairs
  • a sequencing read is only 750 bp - very short fragment
  • must have multiple reads of any one segment because DNA polymerase sometimes makes mistakes
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38
Q

How is overlapping termed when sequencing a genome?

A

-6x coverage = every nucleotide is present in 6 different reads

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39
Q

Who was the human genome project a race between?

A
  • Craig Venter - private company Celera, shotgun sequencing

- Francis Collins - NIH, hierarchical shotgun sequencing w/ genome map

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40
Q

Who and on what was shotgun sequencing first tested?

A

Craig venter first tested this process on a bacterial cell called Haemophilus influenzae (1.8 million bp)

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41
Q

What is the process of shotgun sequencing?

A
  • Extract DNA from bacterial cells
  • Sonicate it
  • Run DNA sequences on a gel
  • Extract the sequences 1.6-2 kb in size
  • Insert the sequences into plasmids to create a clone library
  • Assemble short reads into contigs
  • Attempt to close gaps between contigs by designing primers for the ends and running on PCR to see if a product is produced
  • Generate a second clone library with a different vector
  • Probe a second clone library with pairs of oligonucleotides - if a band forms they have been joined
  • Sequence the product
42
Q

What confirmed that shotgun sequencing works?

A

in 1995 Craig Venter’s group published the full genome of the Haemophilus Infuenzae

43
Q

What are the pros and cons of shotgun sequencing?

A
  • pro - doesnt require a genome map

- con - hard to sequence through repeitive DNA sequences

44
Q

What is sonicating?

A

breaking DNA up into fragments of various sizes by exposing it to a high frequency sound that induces fragments

45
Q

What are contigs?

A

different, non-overlapping portions of the genome used in shotgun sequencing

46
Q

How do you close a gap between contigs in shotgun sequencing?

A

design primers fo the ends and see if a product is produced on the cloned DNA fragment

47
Q

What is the process of hierarchical shotgun sequencing?

A
  • First prepare a clones library of large DNA fragments 300kb (1.6-2kb) - clone into BACs
  • Next probe clones for a particular sequence and identify all the clones with a particular sequence
  • Use chromosome walking, probe for the next contig in a sequence (to find clone contigs)
48
Q

What are overlapping, large fragments of DNA called?

A

clone contigs

49
Q

When was the first publication of the human genome?

A

Feb 15 and 16 Science and Nature

50
Q

What did we learn from the human genome project?

A

complexity doesnt come from the number of genes but rather when genes are tuened on and how theyre spliced

51
Q

What percent of our genome doesnt encode for genes?

A

98%

52
Q

What strange genes were identified through the human genome project?

A
  • ancient viral genomes were retained

- psuedogenes - genes that were once functional, but became mutated or inactivated

53
Q

What is an Alu sequence?

A

a 300bp sequence that reappears millions of times in our genome

54
Q

What percent of our genome is similar to chimpanzees?

A

96%

55
Q

What are some differenced between chain termination sequencing vs next generation sequencing?

A

1) Don’t need to create a clone library - create a DNA library
2) Sequence all DNA fragments at the same time
“massively parallel”
3) Reads are shorter - 300bp

56
Q

What are the steps of NGS?

A
  • Genomic DNA is sonicated into small fragments
  • Adaptors are ligated onto DNA fragments
  • DNA is denatured into single strands and attached to a chip
  • PCR reaction is carried out on the entire chip (Glass slide method)
  • Reversible terminator nucleotides are used to sequence PCR products
  • Add nucleotides one at a time, they glow, and a camera takes an image, then they are removed
  • This last step is repeated over and over again and millions of clusters may be sequenced at once
57
Q

What can be used instead of a chip in NGS?

A
  • a bead could be used instead of chips
  • to do this attach beads by strptavidin-biotin linkages
  • create an oil emulsion with one bead that has one DNA molecule attached to it
58
Q

What is illumina sequencing?

A
  • A form of NGS
  • DNA sequencing reads are short, about 300 bp in length
  • 2,000mb of DNA sequences
59
Q

What is type 1 sequencing?

A
  • A form of NGS
  • Add bases sequentially and degrade whichever bases are not incorporated
  • Camera watching, captures chemiluminescence
  • Reads up to 1000 bp
60
Q

What is solid sequencing?

A
  • A form of NGS
  • Relies on hybridization rather than DNA synthesis
  • 1024 5-mer oligonucleotides used
  • can’t individually label all of them differently so this process must be repeated
  • very accurate, but can’t do it for long sequences of reads
61
Q

What is a newer strategy of NGS (techonolgical advance)

A
  • camera that keeps up with the speed of replication

- watches fluorescently tagged nucleotides be incorporated

62
Q

What is a strategy of NGS that allows you find composition of a DNA strand without replicating it?

A
  • Nanopore with helicase above, how ions flow through the nanopore is impacted by the bases
  • Allows you to directly read the sequence of DNA without repicating it
63
Q

What are histones?

A

special proteins which wind DNA sequences, positively charge (binds negative DNA)

64
Q

What are the histones?

A

There are 8 histones: 2 H2A, 2 H2B, 2 H3 and 2 H4

65
Q

What is a nucleosome?

A

histone + DNA

66
Q

Describe how histones and DNA make up the nucleosome?

A
  • 140-150bp of DNA is wound around histones- makes up the nucleosome
  • 50-70 bp is linker DNA
67
Q

What do nucleosomes form?

A

30nm chromatin fiber

-solenoid or helical ribbon model

68
Q

What are some components of a chromosome?

A

Chromatid - an arm, telomere, centromere

69
Q

What is a karyogram?

A

a depiction of all of the chromosomes of a particular species

70
Q

How are chromosomes numbered?

A

by their size (1 largest - 22 smallest)

71
Q

What are macro and microchromosomes?

A

Macrochromosomes (ours) longer than 50mb, microchromosomes shorter than 20mb

72
Q

What is a holocentric chromosome?

A

multiple structures that act like centromeres

73
Q

What are some staining techniques used to produce chomosome banding patterns/

A

G-banding, R-banding, Q-banding, C-banding

74
Q

What was used to analyze chromosomes before sequencing/

A

staining

75
Q

What are 3 strategies used to count genes?

A

Bioinformatics, homology, and transcript mapping

76
Q

What is bioinformatics?

A

-computational analysis of a genome sequence

77
Q

Describe the process of bioinformatics.

A
  • Search for open reading frames (ORFs)
78
Q

What is an open reading frame? How can you filter for open reading frames? What is a challenge when searching in Eukaryotes?

A
  • DNA sequences that likely encode for a protein, look for ATG ->TAG or other termination codon
  • There are 6 possible reading frames when looking at a sequence: 3 fwd,3 rev
  • Can use length to filter
  • Splicing in Eukaryotes complicates ORF scanning
79
Q

What is a consensus sequence in ORF scanning?

A

sequence that shows the most frequent nucleotide at each position - allows known/likely splice sites to be projected

80
Q

What are some strategies for ORF scanning in Eukaryotes?

A
  • Look for Exon-Intron boundaries
  • Codon bias - look at popular codon choices
  • Upstream regulatory sequences (CPG Islands w high GC content upstream of ORFs)
81
Q

What is an alternative strategy for ORF scanning?

A

Can also scann for non-coding RNA, and this may be helpful in elucidating the structure of some of the RNAs

82
Q

How does homology allow us to count genes?

A
  • all living organisms descend from a common ancestor
  • Homology search in other species
  • “zoo blot” -run genomes of many species on a gel
  • use a radioactive DNA probe to reveal whether or not the sequence of interest is present in other species
83
Q

What genes are conserved between species?

A
  • Exons (coding part of the genome) are highly conserved

- Introns (intergenic/between genes) are less conserved, more variable between species

84
Q

What is transcript mapping? What techniques does it rely on?

A
  • If an ORF encodes a protein, we should be able to isolate complementary mRNA
  • A Northern blot is used to detect the presence of mRNA
85
Q

Describe a Northern blot. What is it used for?

A
  • Used to search for mRNA in transcript mapping
  • DNA sequence of interest is turned into a radioactive probe
  • RNA is extracted from cells, run on denaturing agarose gel electrophoresis, blotting/northern hybridization w DNA probe, DNA probe hybridizes to a single RNA transcript
86
Q

What are bioinformatics, homology, and transcript mapping used for?

A

Used to confirm that a gene sequence has a protein product

87
Q

What is RACE?

A

Rapid Amplification of cDNA Ends

-used to located the end of a coding sequence of a gene

88
Q

Describe the process of RACE.

A
  • A primer is annealed to an RNA strand
  • cDNA synthesis occurs with reverse transcriptase
  • Denature and remove cDNA strand from RNA template
  • Add As to the end with terminal transferase
  • Anneal an anchor primer (with complementary Ts)
  • Second strand synthesis w/ Taq polymerase
  • Continue for standard PCR
  • Sequence PCR reaction - will now include 5’ end of gene
89
Q

How dense is the human genome compared to other species?

A

Very few genes compared to other species

90
Q

Are genes evenly distributed over chromosomes?

A

No, Gene deserts exist sequences of DNA with low density of genes

  • chromosome 13 has density of 3/mb
  • chromosome 19 has a gene density of 22/mb
  • not many genes are present in the region close to the centromere
91
Q

What are some other components that make up chromosomes other than coding DNA?

A
  • LTR -long terminal repeats
  • SINES - short interspersed nuclear element
  • LINES - long interspersed nuclear elements
  • transposons - filler DNA not encoding for genes
  • Exons - proteins coding - take up very little space
92
Q

Describe the trends in gene organization and variation.

A
  • There is an overall trend that as organisms go from simpler to more complex the genome size goes up, however there is also a great deal of variation between organisms
93
Q

How is splicing different in humans?

A

Alternative splicing - occurs in our cells more frequently than in other organisms
Involves joining exons in different ways, 75% of human genes are alternatively spliced

94
Q

What are gene families?

A

groups of genes of identical or similar sequences

95
Q

What is an example of a gene family?

A
  • the globin family - hemoglobin
96
Q

What is a pseudogene?

A

a sequence of DNA that resembles a genuine gene but does not encode a functional RNA or protein

  • may be duplicated - one or several members of the family are non-functional
  • may be unitary - no family members to compensate
97
Q

What are examples of tandemly repeated DNA?

A

minisatellites, microsatellites

This DNA is repeated immediately in a row

98
Q

What are genome browsers and what are some examples?

A

Genome browsers are software packages that display annotation of genomes

  • Genbank -by NIH
  • Ensembl -Sanger Institute
  • UCSC Genome browser - UC Santa Cruz
99
Q

What are examples of interspersed repeats?

A

SINES, LINES, LTRs, and transposons

- interspersed throughout the chromosomes

100
Q

What are two types of repeats in chromosomes?

A

tandemly repeated DNA - like a tandem bicycle (back to back)

interspersed repeats - throughout chromosomes

Genomics (4 decks)

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