Molecular and Mendelian Genetics Exam 2

how to map position of mutation

measuring recombination frequency between mutation and known genes/positions

what limits genetic linkage mapping in humans and many other organisms

few offspring, no controlled crosses, and a low number of visible phenotypes to use in mapping

DNA sequence markers

known sequence variation

VNTR (variable number tandem repeat)

type of sequence  variation; a short DNA sequence repeated several times in tandem

different alleles differ in the number of repeats. 

detectable by PCR amplification followed by gel electrophoresis

Polymerase chain reaction (PCR)

a technique for amplifying a specific DNA sequence from a DNA template (typically a complex mixture of DNA, ie genomic DNA)

Steps of PCR

1. Denaturing of DNA by heating (95 deg. C)

2. Primer annealing (45-68 deg C); primers are designed to be complementary to the target DNA sequence

3. Primers are extended (72 deg C)

4. The cycle is repeated. In each cycle, target DNA region is doubled (amplification of target DNA to 2^n copies where n=# PCR cycles)

Electrophoresis

A technique for the separation of a mixture of molecules by molecular weight, shape, or charge

gel electrophoresis

common technique for separating DNA mixtures by molecular weight (essentially, length in bp) and shape

steps to gel electrophoresis

1. DNA loaded into a well at one end of the gel

2. current applied across gel (in buffer containing ions) pulls DNA toward positive pole

3. gel matrix (agarose, polyacrylamide) acts as sieve

Migration distance proportional to DNA length (in bps) and topology: smallest and most compact fragments migrate the fastest.

DNA is typically visualized with a dye that fluoresces when bound to nucleic acids.

SNP (single nucleotide polymorphism)

type of variation most commonly used in mapping experiments; may be located within genes, but most aren’t; usually not causative for any particular trait. Most are biallelic.

odds ratio

compares the likelihood of observing an outcome under two diff hypotheses

OR = P(outcome A given L)/P(outcome A given no L)

outcome A = observed number recombinant and parental offspring 

L = linkage 

hypothesis may be accepted over the other if the OR is significantly different from 1. 

lod/ log of the odds ratio

commonly used statistical measurement in genetic linkage mapping experiments 

lod = log10 (P(observing outcome, given that the genes are linked with estimated RF)/P(observing outcome, given that the genes are unlinked))

if lod>0, P(observing outcome if linked)>P(observing outcome if unlinked

typical threshold of sig cutoff is lod>3

What is linkage mapping most effective for

mendelian traits

Maximum lod score

has the best value of r; if it does not cross the threshold you dont have statistical significance

GWAS (Genome-Wide Association Study)

a method for locating causative genetic variation for a trait of interest; detects association between the trait and inheritance of SNPs at known genomic locations; performed of a population of unrelated individuals

what does linkage mapping detect

linkage between alleles of two different loci; detected in families

Linkage disequilibrium

when alleles of two different loci are found together in a population at higher than expected frequency; detected by GWAS

linkage equilibrium

when there is no tendency for certain alleles to be inherited together

steps of GWAS

1. define study population

2. for each individual: determine phenotype of interest, determine genotype at mapping SNPs distributed throughout the genome

3. statistically test for association between each SNP and the trait

SNPs in linkage disequilibrium with causative variation will show statistical association with the trait

Manhattan plot

used to display GWAS results

common disease common variant hypothesis

common diseases (or other traits of interest result from alleles of multiple genes, present at high frequency in populations, and each with a small effect on phenotype

expected properties of the genetic material

1. must be easy to replicate

2. must be sufficiently complex to encode phenotypic traits

3. must be highly stable to account for reproducible generation of traits, yet also variable to account for evolutionary change

deoxyribonucleotides

composed of nitrogenous base and deoxyribose sugar; bases have unique combo of chemical groups (=O, -NH2, -CH3); compose DNA; covalently liked

DNA backbone

Consists of repeating sugar and phosphate groups; adjacent sugars joined by phosphodiester bonds. the sequence of nitrogenous bases is unrestricted. is directional.

5’ end

has a free phosphoryl group linked to the terminal 5’ carbon

3’ end

has a free hydroxyl group linked to the 3’ end

bond between the bases

H-bond

general double helix structure

backbone on outside of helix, bases on the inside, complementary strands, antiparallel, most common structure is B-form DNA

B-form DNA

right hand helix, regular spacing (10.5 bp per helical turn), major and minor grooves

cause of major and minor grooves

arise from the angles at which the bases protrude from the backbone

cause of helical structure distortion

complementary bps all have similar geometry, which permits the regular helical shape; mismatched bps have a different geometry; repair enzymes “scan” backbone to detect this

denature

double-stranded nucleic acids become single stranded (a reversible reaction)

renature/hybridize

become double stranded (a reversible reaction); sequence specific

topoisomers

DNA of the same sequence existing in different forms; differ in linking number. can only occur in topologically constrained DNA

linking number

the number of times each strand is wound around the axis of the helix

Topologically constrained DNA

two strands are not free to rotate around each other

relaxed DNA

lowest energy form; for B-form DNA, strands are would around one another once per ~10.5 bps

Supercoiled DNA

strands are either over or underwound compared to relaxed DNA (positively or negatively); most organisms store their DNA in negatively supercoiled form

Positively supercoiled DNA

overwound; has >1 turn per 10.5 bp

negatively supercoiled DNA

underwound; <1 turn per 10.5 bp

topoisomerases

enzymes that change the linking number of a DNA molecule; essential during DNA replication and to maintain/change chromatin organization

chromosomes

what DNA is packaged into in cells

traits of diploid Eukaryotic chromosomes

linear chromosomes, two copies of each

chromosome number is invariant within species

chromosome number varies in different species

highly condensed during metaphase 

less condensed during interphase, where chromosomes occupy relatively fixed territories within the nucleus 

traits of prokaryotic DNA

some have circular chromosomes, some have linear chromosomes

most are haploid

many also have extrachromosomal DNA in plasmids

in situ hybridization

a hybridization-based method for visualizing spatial distribution of specific nucleic acid sequence within cells

steps to in situ hybridization

1. prep sample

2. denature DNA in sample

3. incubate sample with labeled probe = anneal, THEN wash away excess probe

4. detect probe

FISH (fluorescent in situ hybridization)

can be used to detect specific DNA sequences on chromosomes

Chromosome painting

A technique used to visualize the entire chromosome via FISH using multiple probes to chromosome-specific sequences. 

centromere

a specialized region on the eukaryotic chromosome that attaches to the spindle

q

longer chromosome arm

p

shorter chromosome arm

chromatid

one half of a chromosome; sister chromatids only present after s-phase

telomere

a specialized region at the end of linear chromosome that protects DNA ends from damage and from the “end replication problem”

metacentric

p = q

submetacentric

q>p

acrocentric

p has satellites

telocentric

no p arm, not in humans

heterochromatin

darker bands from staining chromosomes with DNA dyes; composed of condensed DNA

euchromatin

lighter bands from staining chromosome with DNA dyes; regions where DNA is less condensed 

cytogenic map

map that shows pattern of banding from stained chromosomes

chromatin

a mix of nucleic acids (DNA and RNA) and protein; make up eukaryotic chromosomes

histones

the major protein component of chromatin; small basic (positively charged) proteins that bind tightly but non-covalently to DNA via electrostatic interaction

5 histones

H1, H2A, H2B, H3, H4

effects of chromatin compaction

allows DNA of the genome to fit inside the nucleus in an organized way; varies across the cell cycle, is critical for regulating gene expression

nucleosomes

first order of DNA condensation; contains a nucleosome core (histone core) particle (2 of each histone except H1) and 146 bps of DNA wrapped around the nucleosome core. called the 10nm fiber, and has a “beads on a string” structure

Histone H1

facultative part of the nucleosome (not present on all nucleosomes) and serves as a clamp that allows DNA to more tightly wrap.

linker DNA

DNA between nucleosomes; length is relatively invariant, about 30-50 bps in mammals

how do histones interact with DNA

Bind DNA in the minor groove, bind non-covalently via electrostatic interaction with backbone, binding is not sequence specific, binding can be moved on DNA strand

30nm fiber

result of twisting 10n, fiber into a “solenoid” structure; requires histone H1 to form

scaffold protiens

non-histone proteins that condense 30nm fiber into 300nm fiber

position effect

the effect of chromatin condensation in a particular genomic position on gene expression

large scale chromosomal mutations

generated by processes including DNA damage and repair, and by unequal crossing over between homologous chromosomes during meiosis; can become fixed in a species and contribute ot genome evolution

terminal deletion

involves loss of a chromosome end (large scale mutation)

can be visualizes by FISH

interstitial deletion

involves loss of a region within a chromosome (large scale mutation)

can be visualized by FISH

duplication

involves duplicating an internal portion of a chromosome (large scale mutation)

can be visualized with FISH

inversion

involves flipping a portion of a chromosome; two types (lsm)

paracentric inversions

inversions that do not include the centromere (lsm)

pericentric inversions

inversions that do include the centromere (lsm)

translocation

involves exchange of material from one chromosome to another; two types; can generate inviable gametes because non-homologous chromosomes can pair at meiosis (lsm)

unbalanced translocations

involve exchange of material from one chromosome to a non-homologous chromosome (lsm)

balanced translocations

involve reciprocal exchange of material between two non-homologous chromosomes (lsm)

how duplications and deletions produce phenotypes

copy number (dosage) of genes within the affected region is altered

how inversions and translocations produce phenotypes

segregation of homologous chromosomes can be altered, leading to inviable gametes and thus infertility

synteny

conserved order of genes linked together on a chromosome, observed in species that share a common ancestor

nondisjuntion

failure of homologous chromosomes or sister chromatids to properly disjoin (segregate) during meiosis or mitosis; leads to aneuploidy

aneuploidy

an altered number of chromosomes in the daughter cell; alters gene dosage; most species are highly intolerant due to the sensitivity of their developmental pathways to gene dosage. plants are tolerant

polyploidy

condition where the complete set of chromosomes is present in more than 2 copies. plants are tolerant, many crops are this (increases fruit size)

two mechanisms for polyploidy

autopolyploidy and allopolyploidy

autopolyploidy

generated by nondisjunction; one individual can cause polyploidy

allopolyploidy

generated by cross-species hybridization followed by nondisjunction

composition of the human genome

mostly intergenic

pyrimidine

single ringed nucleotide (thymine and cytosine)

purine

double ringed nucleotide (adenine and guanine)

substrate for DNA polymerase enzymes

a primed, single stranded template

DNA polymerase enzymes

catalyze synthesis of new DNA strands; synthesize from 5’ to 3’ by adding nucleotides to the 3’ end of the primer; are template-directed enzymes.

The DNA synthesis reaction

Phosphodiester bond formation occurs between the alpha-phosphoryl group on the incoming nucleotide and 3’ OH on the growing strand

beta and gamma phosphates are released as pyrophosphate 

Sanger sequencing

low-throughput sequencing technique; generates one sequence at a time

expensive and slow but accurate 

used in original genome sequencing projects, including the human genome project 

useful if you want to sequence a few short sequences

relatively low number of samples can be processed at a time

a large amount of template DNA and synthesized products are needed to produce detectable flouescence

Next generation sequencing (NGS)

high-throughput sequencing technique, generate many sequences in parallel

most commonly used type now for large-scale sequencing projects 

produces many short sequences (100-150 bp) that must be computationally assembled 

often used in SNP sequencing and in RNA-seq technologies

often used in genome sequencing

relies on tech to amplify DNA templates on a  chip to amounts needed for a detectable synthesis reaction and reversible chain termination chemistry

works with low amount of starting template

third generation sequencing

high-throughput sequencing technique, generates many sequences in parallel

expensive and less accurate 

produces long reads (thousands to tens of thousands of bps)

enables sequencing through repetitive regions and large chromosomal changes that can be impossible to computationally assemble with short reads

massively parallel

done on chips with millions of wells all sequenced in parallel

types of DNA sequencing technologies

sanger sequencing, NGS, third gen sequencing