genetics exam 2 (chapter 6, 7, 20)

Whole genome shotgun sequencing

-Bypasses physical mapping -DNA from entire genome was sheared into both small and large fragments -Cloned into vectors -Insert is sequenced at both ends -Computer identifies overlap

in situ hybridization

-Can locate the position of a gene at a particular site within an intact chromosome -Used to map locations of genes or other DNA sequences within large eukaryotic chromosomes -Use probe to detect "target" DNA -FISH

Hierarchical shotgun sequencing

-Clones large fragments -Inserted into BAC or YAC -Broken into smaller pieces -Cloned into plasmid vectors -Sequence of 500-1000bp from each piece was determined -Computer programs into one long sequence

linked genes

-If there is a large x2 value -Will have higher proportion of combinations of traits found in the parental generation as all three genes are located on the X chromosome and therefore transmit as a unit

LOD score method

-Lod score = log10(probability of certain degree of linkage / probability of independent assortment) -RFLP map: genetic map composed of many RFLP markers -Used to locate genes along particular chromosomes

PFGE

-Method can separate individual chromosomes or large pieces of chromosomes or large pieces of chromosomes -Utilizes alternating pulses of electric current at different angles to each other -Can be used to purify large pieces of DNA for construction of libraries and can be used to identify species or strains of same species -Shotgun sequencing

shotgun sequencing

-Method where small DNA fragments are randomly generated from larger pieces and sequenced -Regions of overlap are used to map fragments -Does not use restriction enzyme of PCR

Southern Blotting

-Must know the sequence as the probe identifies a specific gene

Microsatellites (STR)

-Short, repetitive sequences -Abundantly dispersed throughout a species' genome -Variable in length among different individuals -PCR amplification of particular microsatellites provides an important strategy for analysis of pedigrees

Techniques used in physical mapping

-cloning -chromosome library -chromosome walking -positional cloning -PFGE - YAC, BAC, PAC -hierarchical shotgun vs whole genome shotgun

FISH

-fluorescence in situ hybridization · Fluorescently labeled DNA probe · Probe only binds to specific sequence · Results are compared to the Giemsa-stained chromosomes and the location of a probe can be mapped relative to the banding pattern

RFLP

-recognizes specific DNA sequences and cleave the DNA at those sequences -Individual 3 is heterozygous because it can be cut at 1500bp and has no insertion at 1500bp -DNA samples containing all chromosomal DNA would be isolated -EcoRI digestion would yield so many fragments that the results would be very difficult to analyze (PCR) -Same restriction enzyme is cutting the genomes -Likelihood of linkage between 2 RFLPs is determined by LOD (logarithm of odds) score method

SNP

-single-nucleotide polymorphism -Site in genome where single nucleotide is polymorphic among different individuals

Chromosome walking

A DNA mapping technique that begins with a gene or other sequence that has already been cloned, mapped, and sequenced and "walks" along the chromosomal DNA from that locus, producing a map of overlapping restriction fragments. -only PCR no cloning

Creighton and McClintock's experiment results

Determined that crossovers occur between genes that involve a physical exchange of segments between homologous chromosomes

Uses of genetic maps

Determines that linear order of linked genes along the same chromosome

PAC

P1 artificial chromosome

Genetic linkage definitions

Phenomenon that genes close together on a chromosome tend to be transmitted as a unit, which influences inheritance patterns (not independently assorting)

What is STS

Sequence-tagged site: describes any molecular marker that is found at a unique site in the genome and is amplified by PCR

Synteny

Two or more genes are located on the same chromosome and are physically linked

Fertility plasmid

allow bacteria to mate each other

BAC

bacterial artificial chromosome

Competent cells

bacterial cells able to take up DNA

Competence factors

carry genes that encode proteins that facilitate the binding, uptake, and subsequent incorporation of DNA into bacterial chromosome

complementation - different gene

complementation occurs because coinfected cell makes normal amounts of both genes and will produce plaques

resistance plasmid

confer resistance to antibiotics or toxins

e. coli k12l rII phages

could not produce plaques

degradative plasmid

enable the digestion of unusual substances

col plasmid

encode colicines which are proteins that kill other bacteria

Functional genomics

examines how interactions of genes produces the traits of an organism

independently assorted genes

follow 9:3:3:1

Chisquare analysis with respect to genes linked or independently assorted

if chi square value is very large (deviation between observed and expected values are very large), therefore we reject the hypothesis that the genes assort independently and accept the hypothesis that the genes are linked

Complementation- same gene

no complementation occurs because coinfected cell is unable to make normal product of gene and will not produce plaques

VNTR (minisatellite)

site in genome that contains many repeat sequences

Proteomics

study of all proteins encoded by the genome and their interactions

Horizontal gene transfer

transfer of genes into an organism which is not the offspring of the donor

Virulence plasmid

turn bacterium into pathogenic strain

YAC

yeast artificial chromosome

Calculate gene distance using T. T. Wu equation

· Cotransduction frequency = (1-d/L)3 · D = distance between 2 genes in minutes (what you calculate) · L = size of transduced DNA in minutes

Trihybrid crosses-problems on gene order, calculate gene distance and interference

· Cross 2 true breeding strains that differ with regard to three alleles · Perform a testcross by mating f1 female heterozygotes to male flies that are homozygous recessive for all 3 alleles · Determined f2 generation phenotypes (parental, recombinants) · In crosses with linked genes, parental phenotypes occur most common, double crossovers occur least frequently, and single crossovers occur with intermediate frequency · Calculate the map distance between pairs of genes · Construct the map · Interference: predicts the likelihood of a double crossover from the individual probabilities of each single crossover · Lower than expected value = positive interference · First crossover decreases the probability that a second crossover will occur nearby

Definition of mitotic recombination

· Crossing over during mitosis is rare, so when it does happen, it may produce a pair of recombinant chromosomes that have a new combination of alleles · Causes a twin spot: places in which 2 adjacent regions were different from the rest of the body and from each other

Lederberg and tatum experiments-conclusions

· Discovered genetic transfer in bacteria · Found that e coli had different nutritional growth requirements: · Auxotrophs: cannot synthesize a needed nutrient · Prototrophs: cannot make nutrients from basic components · Met- bio- thr+ leu+ thi+ (produced thr, leu, thi, and lacked met, bio) · Met+ bio+ thr- leu- thi- (produced met, bio, lacked thr, leu, thi) · Discovered that by mixing 2 strains together it produced met+ bio+ thr+ leu+ thi+ (genetic transfer)

Lederberg and Zinder experiment on transduction-conclusions

· Discovered transduction · Used salmonella strains: · LA-22: phe- trp- met+ his+ · LA-2: phe+ trp+ met- his- · Mixed strains but found very cells so they used the U-tube · One side of U-tube had LA-22 and the other was LA-2 · Found that LA-22 side had many plaques on agar while LA-2 had none · LA-22 was the acceptor and LA-2 was the donor

Fig 7.9 calculate gene distance in minutes in linear order of gene transfer

· Distance between genes is determined by comparing their times of entry during interrupted mating experiment

Calculate cotransduction frequency, followed by which gene is closer, see all practice problems

· Frequency = recombinants / total x 100 · Frequency will be high for genes that are close together · Will be very low for distant genes

Using recombinants to determine recombination frequency

· Intragenic recombination produces an equal amount of recombinants · ½ are wild-type · ½ are double mutant · However only wild-type is detected in the infection of e. coli k12l, so total number of recombinants must be multiplied by 2 · Frequency of recombinants = 2[wild-type plaques obtained in e. coli k12l] / total number of plaques

cloning

· Isolate the DNA either by PCR or restriction enzyme · Clone the DNA into a plasmid · Plasmid will transform into a bacterial cell · DNA can then be screened

map distance

· Map distance = number of recombinants / total number of offspring x 100 · Measured in map units (mu) or centimorgans (cM)

Homoallelic mutants

· Mutations that happen to be located at exactly the same site in a gene and are not able to produce any wild-type recombinants so the map distance would be 0

Calculation of gene distance from gene to centromere in octad and tetrad

· Ordered: ascus is very tight, preventing spores from randomly moving around · FDS: 4:4 ratio · SDS: 2:4:2 or 2:2:2:2 ratio · Map distance = (0.5)(SDS) / total x 100 · Unordered: ascus provides enough space for spores to randomly mix together · PD ascus: contains 100% parental · T ascus: contains 50% parental and 50% nonparental · NPD ascus: contains 100% recombinant · map distance = (T + 6NPD) / total x 0.5 x 100

Difference between RFLP and microsatellites

· RFLPs used restriction enzymes and southern blots · Microsatellites use PCR since there are repeated sections and it would cut too many times with RE

Problems on linear order or interrupted mating-experiment 7A on interrupted mating conclusions

· Rationale: the time it takes genes to enter the recipient cell is directly related to their order along the bacterial chromosome · The Hfr chromosome is transferred linearly to the F-recipient cell · Interrupting mating at different times would lead to various lengths being transferred and the order of genes along the chromosome can be deduced by determining the genes transferred during short matings vs those transferred during long matings

Chromosome library

· Sample of chromosomal DNA can be digested into many smaller pieces with restriction enzymes · Fragments will then be cloned into vectors to create a chromosome-specific library

Limitations of map distance calculations in Sturtevant's data

· Showed an inaccuracy because he did not account for multiple crossovers · When there is a large distance between 2 genes, the likelihood of multiple crossovers increases · The observed number of recombinant offspring tends to underestimate the actual distance between genes · Far distance = increase in chances of double crossovers

Bernard Davis experiments-conclusions

· Showed that bacterial strains must make physical contact for the transfer of genetic material to occur (conjugation) · Used a U-tube which contains a filter at the bottom which has pores that were large enough to allow the passage of genetic material but small enough to prevent the passage of bacterial cells · Placed 2 strains on opposite sides of the filter and applied pressure/suction to promote the movement of liquid through filter · Determined that without physical contact, the two strains did not transfer genetic material to each other, no bacteria were seen on the plates

Cistron

· Smallest genetic unit that gives negative complementation test · If 2 mutations occur in the same cistron, they cannot complement each other

positional cloning

· Strategy to clone a gene based on its mapped position along a chromosome · Common form is chromosome walking (no cloning involved)

Conclusions from Morgan's experiment

· The genes for body color, eye color, and wing length are all located on the X-chromosome and will be inherited together · Due to crossing over, homologous X chromosome in the female can exchange pieces of chromosomes to create new combinations of alleles · Likelihood of crossing over depends on the distance between two genes meaning crossing over is more likely to occur between two genes that are far apart from each other

Deletion mapping

· Used to localize many rII mutations to a fairly short region in gene A or gene B

e. coli B rII phages

· produce large plaques that have poor yield of bacteriophages as it lyses so quickly that it does not have time to produce new phages

e. coli k12s rII phages

· produce normal plaques that gave good yield of phages

Techniques used in linkage mapping

· relies on genetic crosses -Frequency of recombinant offspring to map genes -Molecular marker: DNA segment that is found at a specific site and can be uniquely recognized -Distance between linked molecular markers can be determined from the outcome of crosses -genes are mapped relative to each other -in map units or centiMorgans -RFLP -southern blotting -microsatellites (STR) -SNP -minisatellite (VNTR)

Techniques used in cytogenetic mapping

· relies on light microscopy -Commonly used in eukaryotes which have much larger chromosomes -Use Giemsa stain for chromosome bands -genes are mapped relative to band locations on chromosomes -Tries to determine the location of a particular gene relative to a banding pattern -Used as first step in localization of genes in plants and animals -In situ hybridization

Vertical gene transfer

· transfer of genes from mother to daughter cell or from parents to offspring