Genetics Exam 3 Dr. Paris

Polymorphism

the existence of two or more differences in DNA sequence

Does a wild-type human genome sequence exist?

no

Why don't most polymorphisms influence phenotype?

Because codons make up less than 2% of the human genome, and many mutations in codons dont change the amino acid

How do many deleterious mutations disappear from the population?

Through natural selection

4 categories of genetic variation

1. Single nucleotide polymorphisms

2. Deletion-insertion polymorphisms (DIPs)

3. Simple sequence repeats (SSRs or microsatellite)

Single nucleotide polymorphisms

•One base pair changes

Deletion-insertion polymorphisms (DIPs)

•Short insertions or deletions of a single or a few base pairs

Simple sequence repeats (SSRs or microsatellite)

•1-10 base sequence repeated 15-100 times in tandem

Copy number variants (CNVs)

•large blocks of duplication or deletion with population frequency of < 1%

What produces new alleles of copy number variants (CNVs)?

Unequal crossing-over

What are CNVs?

Tandem sequence repeats more than 10 bp long. Not a common event, so most CNVs are inherited, rather than being a new mutation.

What does determining genotype depend on?

it depends on isolating a gene and analyzing the alleles.

Polymerase chain reaction (PCR)

•Method of making many copies of a target region of DNA.

•First developed in 1985 by Kary Mullis

•Faster, less expensive, and more flexible way to amplify specific fragments of DNA (compared to molecular cloning)

•Extremely efficient - can amplify DNA from a single cell or from some archaeological samples

What defines the target regions in PCR?

Two oligonucleotide primers.

• One primer is complementary to one strand of DNA at one end of the target region

• The other primer is complementary to the other strand of DNA at the other end of the target region

The three steps in each PCR amplification cycle

(1) Denature strands

(2) Base pairing of primers

(3) Polymerization from primers along templates

What can PCR product size determine?

genotype

What is sickle cell anemia caused by?

• a SNP in the Hb beta gene, chromosome 11 (glutamic acid - GAG valine - GTG).

• PCR genotyping can identify carriers of the disease and homozygous individuals.

Analysis of the Huntington disease locus by PCR:

-Autosomal dominant disorder

-Normal allele has < 34 CAG repeats

-Disease-causing alleles have 42 or more CAG repeats

Can Fetal and embryonic cells can be genotyped using PCR?

Yes, by:

• Prenatal genetic diagnosis

•Genotyping fetal cells

•Cells isolated by amniocentesis—fetal cells in amniotic fluid are extracted using a needle.

• Preimplantation embryo diagnosis

•Utilizes in vitro fertilization and PCR

•Genotype embryos before placing in womb

What kind of PCR is used for DNA fingerprinting?

Multiplex PCR

multiplex pcr

multiple primers for multiple targets

Short hybridization probes can distinguish _______________ mismatches

single-base

Oligonucleotides

Oligonucleotides are short single or double-stranded fragments of DNA or RNA.

How hybridization probes are used on microarrays for genotyping:

• Many Allele-specific oligonucleotides (ASO) are attached to a solid support (like a silicon chip).

• Genomic DNA is used to probe theASO chip (microarray).

• Fluorescent output is proportional to the number of copies of each allele.

Up to ______________ loci can be genotyped simultaneously.

4 million

Object of Positional cloning:

the object is to identify disease-causing genes by genetic linkage to polymorphic loci

Strategy of positional cloning:

• One gene is tracked by phenotype, and one gene is tracked by genotype.

• Microarrays used to analyze millions of two-point crosses that all test for linkage between a disease locus and a DNA marker.

The steps involved in positional cloning

• Region of interest narrowed by finding closely linked DNA markers (markers are close neighbors to the gene).

• Candidate genes are located in the region of interest.

• Sequence and expression of candidate genes are determined in normal and diseased individuals.

Allelic heterogeneity

• disease caused by different mutations in the same gene

Compound heterozygote

Individual with different mutant alleles of the same gene:

•Individuals with certain alleles may respond to drug treatment, while others do not.

Locus heterogeneity

• disease caused by mutation in one of two or more different genes

How much does genome sequencing cost?

$400-$500 (sequencing the whole exome costs even less)

High-throughput sequencing

•Individual DNA molecules are anchored in place

•Each base is identified before the next one is added

•Increased sensitivity eliminates need for cloning or PCR

How can we tell which polymorphism causes a disease?

•Transmission pattern

•Predicted effect on protein function

•Clues from other genome sequences

What is CODIS

Combined DNA Index System, and is the term used to describe the FBI's program of DNA databases. Data can match DNA from crime scene to a person, or can establish innocence.

Chromosomes

• support the packaging, replication, segregation, and expression of genetic information.

• Chromosomes are the separate pieces of chromatin that behave as a unit during cell division

Chromatin

is the generic term for any complex of DNA and protein found in a nucleus of a cell

chromatin makeup:

Chromatin is ~ 1/3 DNA, 1/3 histones, 1/3 non-histone proteins

If stretched out, the total DNA in a single cell would be about _______ long.

6 feet

Histones

• small, positively-charged, and highly conserved

• bind to and neutralize negatively charged DNA

Five types of histones

H1, H2A, H2B, H3, and H4

2 molecules each of H2A, H2B, H3, and H4 sot on top of H1 and make up the ____________

nucleosome core

There are ______ to ___________ molecules of each kind of nonhistone protein that also makes up chromatin

200-200,000

Functions of non-histone proteins

•Structural role - chromosome scaffold

•Chromosome replication - e.g. DNA polymerases

•Chromosome segregation - e.g. kinetochore proteins

•Active in transcription - largest group

the nucleosome

-is the fundamental unit of chromosomal packaging.

• Nucleosomes resemble beads on a string.

how do nucleosomes affect genetic function?

by their spacing and structure

nucleosome structure

• 160 bp of DNA wraps twice around a nucleosome core

• 40 bp of linker DNA connects adjacent nucleosomes

• Histone H1 associates with linker DNA as it enters and leaves the nucleosome core

2 models of higher-level chromatin compaction:

1. nucleosome supercoiling.

2. radial loop-scaffold model.

radial loop-scaffold model

• Several nonhistone proteins (NHPs) bind to chromatin every 60-100 kb and tether the 300 Å fiber into structural loops

• Other NHPs gather several loops together into daisylike rosettes

-Condensins may further condense chromosomes into a compact bundle of many rosettes for mitosis

long chromosome arm is called

q arm

short chromosome arm is called

p arm

Fluorescent in situ hybridization (FISH) is used to characterize genomes

•Chromosomes are spread on a glass slide and denatured to make them single stranded

•A DNA sequence is labeled with a fluorescent tag to make a probe.

•The probe hybridizes to chromosomes at complementary regions.

Karyotyping

is looking at chromosomes under a microscope.

Spectral karyotyping (SKY) is a variation of FISH

probes specific for each chromosome are labeled with a different fluorescent dye

Heterochromatin

highly condensed, usually inactive transcriptionally

constitutive heterochromatin

• condensed in all cells (e.g. most of the Y chromosome and all pericentromeric regions).

• darkly stained regions of chromosomes

facultative heterochromatin

condensed in only some cells and relaxed in other cells (e.g. position effect variegation, X chromosome in female mammals)