1
Q
Module 3
Genetic mapping / linkage analysis
A
- based on the use of genetic techniques, including planned breeding experiments or examination of family histories (pedigrees)
- first method used to map genome
- any genomic feature can be used for linkage map
2
Q
Module 3
Physical mapping
A
uses molecular biology techniques to examine DNA molecules directly in order to identify the positions of sequence features, including genes
3
Q
Module 2
DNA sequencing has one major limitation:
A
- only with the most sophisticated and recently introduced technology is it possible to obtain a sequence of more than about 750 bp in a single experiment
- human genome is 3.2 billion bp so 3.2 billion ÷ 750 = 4,300,000 sequencing runs → underestimate, have to take repeating DNA sequences into account
4
Q
Module 2
shotgun method
A
- The DNA molecule is broken into small fragments
- each fragment (entire genome) is sequenced
- The master sequence is assembled by searching for overlaps between the sequences of individual fragments and assembled into contigs
- markers are used to anchor contigs
- constructing a clone contig map
5
Q
Module 2
shotgun method is the standard approach for genome sequencing, but it suffers from two problems. Name one.
A
- can lead to errors if the genome contains repetitive DNA sequences
- when a genome w/repetitive DNA is broken into fragments, some of the pieces will contain the same sequence motifs
- ease to reassemble so a portion of DNA between the repeats is left out
- or erroneously connect together two separate pieces of the same or different chromosomes
- w/a genome map, If features on either side of a repetitive region match the genome map, then your good
6
Q
Module 2
shotgun method is the standard approach for genome sequencing, but it suffers from two problems. Name one.
A
- genome sequence might be made up of short segments separated by gaps that represent parts of the genome that, by chance, are not covered by the sequences that have been obtained.
- If these segments are unconnected, how can they be positioned correctly relative to one another
- By anchoring the segments onto a genome map, the correct genome sequence can be obtained, even if that sequence still contains some gaps
7
Q
marker-assisted selection
A
- an indirect selection process where a trait of interest is selected based on a marker (morphological, biochemical or DNA/RNA variation linked to a trait of interest (i.e. productivity, disease resistance, abiotic stress tolerance, and quality), rather than on the trait itself.
- possible only if a genome map is available
8
Q
Module 3
DNA markers
A
- Mapped features that are not genes
- must have at least two allele
- anchor clones & contigs
- verify chromosome walking technique
9
Q
Module 3
restriction fragment length polymorphisms (RFLPs)
A
- first type of DNA marker to be studied
- When an SNP is located at a restriction site
- restriction enzyme does not always produce the same set of fragments w/DNA
- some restriction sites are polymorphic, existing as two alleles
- one allele has the correct sequence for the restriction site & is cut by the enzyme,
- 2nd allele’s sequence is not recognized by the restriction enzyme and is cut differently
10
Q
Module 3
two methods for typing an rfLp
A
- RFLPs can be typed by Southern hybridization
- DNA is digested & separated in an agarose gel
- smear of restriction fragments is transferred to a nylon membrane and probed with a piece of DNA that spans the polymorphic restriction site
- If the site is absent, then a single restriction fragment is detected
- if site is present, then two fragments are detected
- RFLP can also be typed by PCR
- primers are used that anneal on either side of the polymorphic restriction site
- After PCR, the products digested & separated in an agarose gel
- If is absent, then one band is seen on the agarose
- if the site is present, then two bands are seen
11
Q
Module 3
Microsatellites aka
- short tandem repeats (STRs)
- GENOME-WIDE REPEATED SEQUENCES
A
- DNA markers
- repeats are usually 13 bp or less
- more popular than minisatellites as DNA markers
- more conveniently spaced throughout the genome
- typically consist of 10–30 copies of a repeat that is no longer than 6 bp in length, and so they are much more amenable to analysis by PCR
12
Q
Module 3
simple sequence length polymorphisms (SSLPs)
two types:
A
- Minisatellites aka variable number of tandem repeats (VNTRs): repeat unit is up to 25 bp in length
- Microsatellites aka short tandem repeats (STRs): repeats are shorter, usually 13 bp or less
13
Q
Module 2
oligonucleotide
A
- a short, single-stranded DNA molecule
- usually less than 50 nucleotides long
- synthesized in the test tube
14
Q
Module 3
simple sequence length polymorphisms (SSLPs)
A
- DNA marker
- arrays of repeat sequences that display length variations
- different alleles contain different numbers of repeat units
- can be multiallelic
15
Q
Module 3
Minisatellites aka
- variable number of tandem repeats (VNTRs)
- tandemly repeated sequences
A
- repeat unit is up to 25 bp in length
- not spread evenly around the genome
- tend to be found more frequently in the telomeric regions at the ends of chromosomes
- Most minisatellite alleles are longer than 300 bp
- cuz they are large and many of them in a single array PCR does not work well w/them
- PCR typing is much quicker and more accurate with sequences less than 300 bp
16
Q
capillary electrophoresis
A
- used to type STRs
- uses Polyacrylamide gels
- have smaller pore sizes than agarose gels and allow greater precision in the separation of molecules of different lengths
- use fluorescence detection
- fluorescent label is attached to one or both of the primers before PCR
- DNA goes past fluorescence detector, read by a computer which reads time of passage & correlates w/a set of size markers identifying precise length of the product
17
Q
Module 3
single-nucleotide polymorphisms (SNPs)
A
- position in a genome where some individuals have one nucleotide (e.g., a G) and others have a different nucleotide (e.g., a C)
- there are vast numbers of SNPs in every genome (approximately 10 million in the human genome)
- In most eukaryotes SNP exist every 10 kbp on average. Human genome, 320,000 SNPs
- some give rise to RFLPs
- originates when a point mutation occurs in a genome, converting one nucleotide into another
- vast majority of SNPs are biallelic
- enable very detailed genome maps to be constructed
- Makes possible highly detailed maps
18
Q
Module 3
Solution hybridization
A
- in wells of a microtiter tray
- uses detection system that discriminates between nonhybridized, single-stranded DNA and the double-stranded product from hybridization
- most popular uses dye quenching
- reporter probe is used to follow product formation during real-time PCR
- dye is attached to one end of the oligonucleotide and the quenching compound to the other end
- Hybridization indicated by generation of the fluorescent signal
- When used this way, dye-quenching is aka molecular beacons.
19
Q
Module 3
an oligonucleotide will hybridize with another DNA molecule only if
A
- the oligonucleotide forms a completely base-paired structure with the second molecule
- it must base-pair 100%
- To achieve this level of stringency, the incubation temperature must be just below the melting temperature, or Tm, of the oligonucleotide. At temperatures above Tm, even the fully base-paired hybrid is unstable
20
Q
Module 3
Oligonucleotide hybridization can discriminate between
A
the two alleles of an SNP
21
Q
Module 3
DNA chip
A
- SNP typing strategies
- glass/silicon wafer of glass or silicon w/area <= 2 cm2 and density of up to 300,000 oligonucleotides/cm2
- has different oligonucleotides in a high-density array
- DNA labeled with a fluorescent marker and pipetted onto chip surface
- Hybridization detected via fluorescence microscope
- positions of fluorescent signal emitted indicates which oligonucleotides have hybridized w/DNA
- indicates which of the two versions of a SNP is present in the test
- can type for both alleles of each SNP
22
Q
Tm
A
- melting temperature in degrees Celsius
- calculated from the formula
- Tm = (4 × number of G and C nucleotides) + (2 × number of A and T nucleotides)
- This formula gives a rough indication of Tm for oligonucleotides of 15–30 nucleotides in length
23
Q
Module 3
- Other SNP typing methods make use of an oligonucleotide whose mismatch with the SNP occurs at its:
- An oligonucleotide of this type will hybridize to the mismatched template DNA with a short _____ _____
A
- extreme 5ʹ- or 3ʹ-end
- non-base-paired tail
24
Q
Module 3
oligonucleotide ligation assay (OLA)
A
- two oligonucleotides anneal next to each other on the DNA w/3ʹ-end of one positioned exactly at the SNP
- if both oligonucleotide base-pair to DNA 100%, the two can be ligated together
- If one does not base-pair to DNA 100%, the two cannot be ligated together
- SNP typed if ligation product is synthesized
- If a single SNP is being assayed, formation of ligation product can be identified by running the postreaction mixture in a capillary electrophoresis system
25
# Module 3
amplification refractory mutation system, or ARMS test
* test oligonucleotide is one of a pair of PCR primers
* If the 3ʹ-nucleotide of the test primer anneals to the SNP, then it can be extended by Taq polymerase and the PCR can take place
* if it does not anneal because the alternative version of the SNP is present, then no PCR product is generated
26
recombination frequency (or rate of recombination)
* a measure of the distance between two genes
* two genes that are close together will be separated by crossovers less frequently than two genes that are more distant from one another
* % of recombinant progeny in a cross
* (recombinants / ttl offspring) × 100
* units: cM (centimorgans)
* the higher the # the more closely genes are physically linked
27
# Module 3
linkage group
A group of linked genes
28
# Module 3
map units aka centiMorgans (cM(
* unit of measurement for distances on genetic maps
* abbreviated m.u.
* one map unit = 1% recombination rate
29
recombination hotspots
* regions of the genome that undergo recombination at significantly higher rates than average and contribute to genomic diversity within and between populations
* present at multiple locations in the human genome
* regions enriched for these hotspots include telomeres, and their neighboring sequences, as well as other regions containing repetitive sequences such as segmental duplications
30
pedigree analysis issues
* Often, only limited data are available
* interpretation is often difficult because a human pairing rarely results in a convenient test cross
* often the genotypes of one or more family members are unobtainable because those individuals are dead or unwilling to cooperate
31
# Module 2
three natural methods that exist for transferring pieces of DNA from one bacterium to another
* transformation
* conjugation
* transduction
32
conjugation
* two bacteria come into physical contact and one bacterium (the donor) transfers DNA to the second bacterium (the recipient)
* the transferred DNA can be a copy of some or possibly all of the donor cell’s chromosome, or it could be a segment of chromosomal DNA, up to 1 × 106 base pairs or 1 megabase (Mb) in length, integrated in a plasmid (called episome transfer)
33
Transduction
transfer of a small segment of DNA, up to 50 kb or so, from donor to recipient via a bacteriophage
34
# Module 3
transformation
recipient cell takes up from its environment a fragment of DNA, rarely longer than 50 kb, released from a donor cell
35
# Module 2
DNA shearing
* an experimental process used to prepare DNA for analysis or other processing
* uses mechanical instruments to randomly cleave DNA
* DNA is sheared to the desired fragment range
36
# Module 2
Contig
* A continuous sequence of DNA assembled from overlapping cloned DNA fragments
* Each contig is a genomic clone, usually in a cosmid or a yac
* its a single clone that is part of a set of overlapping clones
* used in contig mapping
37
# Module 2
clone contig sequencing method
* often used to sequence eukaryotic genomes
* used to obtain the sequence of a genome
* **older method which uses a traditional step-wise approach of sequencing clones that have been placed on a genomic map**
* **markers are used to anchor clones**
* **a set of contiguous clones is sequenced individually**
* **sequence is assembled based on the position of the marker**, constructing a clone contig map
* A library is constructed by cloning large overlapping DNA inserts spanning the entire genome
* These DNA inserts are usually generated by partial restriction and cloned into cloning vectors: BACs, YACs or COSMIDS
* The inserts in the library of clones are assembled into clone contigs on the basis of overlap
* for example, by chromosome wlaking, or clone fingerprinting technique
* Inserts are then organized into a physical map of the genome
38
contiguous
* sharing a common border; touching
* next or together in sequence
39
# Module 3
physical mapping techniques can be be grouped under 2 categories
* Methods that involve direct examination of DNA molecules or chromosomes
* Methods that assign markers to DNA fragments whose positions within an intact DNA molecule are known or can be inferred
40
# Module 3
restriction mapping
* enable the positions of restriction sites to be located in a DNA molecule
* attempts to increase the marker density on a genome map by using an alternative method to locate the positions of some of the nonpolymorphic restriction sites
* more applicable to small rather than large molecules, less than 50 kb in length
41
# Module 3
restriction mapping
second step
* another sample of the molecule is digested with the second enzyme, and the resulting fragments are again sized in an agarose gel
* results so far enable the number of restriction sites for each enzyme to be worked out but do not allow their relative positions to be determined
42
# Module 3
restriction mapping
first step
* sample of the DNA molecule is digested with just one of the enzymes and the sizes of the resulting fragments are measured by agarose gel electrophoresis
43
# Module 3
restriction mapping
fourth step
* A partial restriction is carried out: single enzyme is used but the digestion does not go to completion
* reaction is incubated only for a short time or a suboptimal incubation temperature is used
* Partial restriction leads to partially restricted fragments that still contain one or more uncut restriction sites. The sizes of the partially restricted fragments enable the map to be completed
44
restriction mapping yield an unambiguous map if there are relatively _____ cut sites for the enzymes being used. However, as the number of cut sites \_\_\_\_\_, so also do the numbers of \_\_\_\_\_, \_\_\_\_\_, and ______ products whose sizes must be measured and compared. A stage will be reached when a digest contains so many fragments that individual bands merge on the agarose gel, increasing the chances of one or more fragments being measured incorrectly or missed entirely.
* few
* increases
* single-, double-, and partial-restriction
45
optical mapping
* does not need than electrophoresis
* restriction sites are located simply by observing the cut DNA molecules under a microscope
* DNA molecules are uncoiled and extended into a linear configuration so that the locations of cuts made by a restriction enzyme can be visualized
46
optical mapping:
molecular combing
* new form of optical mapping
* silicone-coated coverslip is dipped into a solution of DNA, left for 5 minutes
* DNA molecules attach to the coverslip by their ends
* coverslip removed from the solution at a constant speed, typically 0.3 mm s–1
* force required to pull DNA through meniscus causes them to line up
* Once in the air, the surface of the coverslip dries, holding DNA as an array of parallel fibers
* restriction sites less than 800 bp apart can be visualized
47
optical mapping:
gel stretching
* earliest form of optical mapping
* Chromosomal DNA was suspended in molten agarose with restriction enzymes
* DNA was placed on a microscope slide held at a slight angle so gel flowed slowly down slide, cooled & solidified
* this made DNA line up and become extended
* enzymes were activated by adding magnesium ions (all restriction enzymes require magnesium in order to work)
* fluorescent dye, such as DAPI is added to DNA for visualization
* restriction sites in the extended molecules become gaps as the degree of fiber extension is reduced by the natural springiness of the DNA, enabling the relative positions of the cuts to be recorded
* has some distortion
48
Molecules over _____ in length are difficult to purify and extend without _____ \_\_\_\_\_, so most optical maps are built up from the data obtained from a series of \_\_\_\_\_fragments. The procedure is labor-intensive, many separate observations have to be made, and the amount of labor increases disproportionately as the length of the starting molecule increases
* 1 Mb
* accidental breakage
* overlapping
49
microfluidic device for optical mapping of restriction sites
* DNA molecule becomes partially extended as it passes through a grid of electrodes
* it is fully extended when it enters the nanochannel, which is only slightly wider than the double helix
* DNA is cut within the nanochannel which contains a magnesium ion gradient so that the restriction enzyme is activated
50
fluorescent in situ hybridization (FISH)
* technique for physical mapping of DNA molecules
* marker used is a DNA sequence contained in the DNA
* sample of dividing cells is dried onto a microscope slide and treated so that the chromosomes become denatured but do not lose their characteristic metaphase morphologies
* marker is flagged w/fluorescent DNA probe that is complementary & binds to the marker
* The position the probe hybridizes to the chromosomal DNA is visualized by detecting the fluorescent signal emitted by the labeled DNA
* identifies the position of a marker relative to the centromere & chromosome bands
51
fluorescent in situ hybridization (FISH)
limitations
* cannot achieve any degree of highresolution mapping because two markers have to be at least 1 Mb apart to be resolved as separate hybridization signals
* Even with new innovations, markers closer than 25 kb cannot be resolved
52
fiberFISH
disadvantage
solution
* use a peptide nucleic acid (PNA) as the probe
* A peptide nucleic acid has an amide backbone instead of the sugar– phosphate structure found in a standard nucleic acid
* ssDNA can attach to two PNAs at the same time
* one by Watson–Crick pairing and one by Hoogsteen pairing.
* The resulting triplex structure, PNA2DNA, is more stable than a DNA–DNA hybrid and is unlikely to break down during the optical mapping procedure
53
fiberFISH
disadvantage
* ensuring probe remains attached to its specific position on the DNA fragment
* w/traditional hybridization probe, the target DNA must be partially denatured to expose a single-stranded region so the probe can anneal
* The second DNA strand competes w/the probe & can displace it, re-forming the double-stranded molecule
* If this occurs prior to passage of the DNA past the detector, then no data will be obtained
54
fiberFISH
* uses purified DNA instead of intact chromosome
* a modified version of optical mapping
* uses stretched DNA fragments in microfluidic devices
* probe can be designed to target any desired DNA sequence
* no limit to the types of marker that can be detected
55
optical mapping
GC-rich regions
* GC-rich regions located by partially denaturing DNA
* temperature is raised or a chemical denaturant used in the microfluidic solvent
* Because a G-C base pair has three hydrogen bonds, compared to just two for A-T, the GC-rich regions are more likely to remain double-stranded
* a double-strand-specific dye is added to reveal GC-rich regions
* GC-rich regions may indicate locations of genes, so this method can be useful when a genome sequence is being annotated.
56
# Module 3
sequence-tagged site (STS)
* short DNA sequence
* between 100 and 500 bp
* easily recognizable
* occurs only once in the chromosome or genome
* assigning markers to genome fragments, on the basis that two markers that occur in the same fragment must be located close to one another in the genome
* most versatile
57
# Module 3
sequence-tagged site (STS)
visual
* fragments span the entire length of a chromosome
* each point on the chromosome present (on average) in five fragments
* The two blue markers are close together on the chromosome map so there's a high probability they will be found on the same fragment
* The two green markers are more distant from one another and so are less likely to be found on the same fragment
58
# Module 3
To qualify as an STS, a DNA sequence must satisfy two criteria:
* its sequence must be known
* PCR assay is set up to test for the presence or absence of the STS on different DNA fragments
* it must have a unique location in the chromosome or genome being studied
* If it occurs more than once, then the mapping data will be ambiguous
* an STS cannot include sequences found in repetitive DNA
59
# Module 3
STS markers can be obtained by
* expressed sequence tags (ESTs)
* SSLPs
* random genomic sequences
60
# Module 3
expressed sequence tags (ESTs)
* short sequences obtained by analysis of complementary DNA (cDNA) clones
* cDNA is prepared by converting an mRNA preparation into double-stranded DNA
* because mRNA is derived from genes, cDNAs and the ESTs obtained from them represent the genes that were being expressed in the cell that produced the mRNA
* a rapid means of gaining access to the sequences of important genes
* valuable even if their sequences are incomplete
* can also be used as an STS, if it comes from a unique gene and not from a member of a gene family in which all the genes have the same or very similar sequences.
61
# Module 3
STS mapping procedure is the collection of DNA fragments spanning the chromosome or genome being studied. This collection is sometimes called the _____ \_\_\_\_\_
mapping reagent
62
mapping reagent can be assembled in two ways
* clone library
* panel of radiation hybrids
63
radiation hybrids
* a cell or organism that contains fragments of chromosomes from a second organism
* Result of irradiation of human cells: the chromosomes break into fragments, with smaller fragments generated by higher X-ray doses.
* A radiation hybrid is produced by fusing an irradiated human cell with an untreated hamster cell
* fragments are 5–10 Mb in size, with each cell containing fragments equivalent to 15–35% of the human genome
* collection of cells is called a radiation hybrid panel
* used as a mapping reagent in STS mapping, provided PCR assay used to identify the STS does not amplify the equivalent region of DNA from the hamster genome
64
flow cytometry
* dividing cells (ones w/condensed chromosomes) are broken open so intact chromosomes are obtained
* chromosomes are stained w/fluorescent dye
* amt. of dye that a chromosome binds depends on its size
* chromosome preparation is diluted and passed through a fine aperture, producing a stream of droplets, each one containing a single chromosome
* droplets pass through a detector measuring amt. of fluorescence identifying droplets w/particular chromosome
* electric charge is applied to droplets containing the desired chromosome to be deflected and separated from the rest
* different chromosomes w/similar sizes are separated by using dye w/a preference for AT- or GC-rich regions
* similar sized chromosomes rarely have identical GC contents & are distinguished by the amt of AT- or GC-specific dye that they bind
65
# Module 3
Two Uses of Genomic Markers
* Arranging DNA clones into their original context in the intact genomepreparation for sequencing.
* Identification of linked genes, e.g. human disease genes (genetic testing), or valuable trait genes (termed marker assisted breeding)
* don't need to know the gene that causes the disease, can use the marker to diagnose
* must be close enough to the gene
Molecular Genetics
flashcards
Decks in class (12)
# Cards
-
Chapter 2: Studying DNA78
-
Chapter 365
-
Chapter 4: Sequencing Genomes39
-
Chapter 5: Genome Annotation74
-
Chapter 6: Identifying Gene Function72
-
Chapter 12: Transcriptomes6
-
Chapter 7: Eukaryotic Nuclear Genomes44
-
Chapter 8: Genomes of Prokaryotes and Eukaryotic Organelles23
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Chapter 10: Accessing the Genome45
-
Chapter 11: Assembly of the Transcription Initiation Complex77
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Chapter 12: Synthesis and Processing of Bacterial RNAs35
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Chapter 13: Synthesis and Processing of the Proteome16
