Genomics 1

Most common microsatellite in humans

(CA)n where n is anywhere from 5-50 repeats

If RFLPs are linked, offspring will result in...

... a much higher percentage of parental types than recombinant types that depends on the distance between the RFLPs

If RFLPs are not linked, offspring will result in...

... a ratio of 1:1:1:1

Process to create a STS

1) Add PCR primers complimentary to the sequences's unique flanking sequences 2) Many cycles of PCR 3)Run on gel to identify length of sequence

Whole-Genome Shotgun Sequencing

1) Bypass physical mapping steps 2)Shear DNA into small and large pieces and clone into vectors (no contig) 3) Determine chromosomal DNA sequence at both ends (double-barrel shotgun sequencing) 4)Based on overlapping regions create one contiguous sequence

General Process of Physical Mapping

1) Chromosome of interest digested with restriction enzymes after chromosome sorting 2) Fragments are cloned into vectors 3) Cloned pieces compared for overlapping and used to construct a contig (continuous region) of the chromosome

Benefits of cosmids

1) DNA can be replicated like a plasmid 2) DNA can be packaged into a protein coat like a phage

Process of Positional Cloning

1) DNA of each chromosome is cut into identifiable, overlapping restriction fragments 2) Cloned in a YAC or BAC for first round normally (although can be PAC or cosmid) 3) Probes identify overlapping ends (Chromosome Walking) 4) Large fragments are cut into smaller fragments which are then cloned an ordered again 5) Results in fragments of about 1000 bp that can be easily sequenced

RFLP Mapping

1) Determine the distance between linked RFLPs 2) RFLPs can be used as chromosomal markers 3) Uses band patterns on gels instead of phenotypes of individuals (as in genetic crosses) to distinguish individuals

How are molecular markers used to identify disease genes?

1) For inherited diseases where the diseased gene is unknown, you can follow the transmission of polymorphic molecular markers in family pedigrees 2) Knowning that patients with a disease also have a specific molecular marker can help the gene be identified through cloning techniques, because the gene is likely close to the marker

Pyrosequencing

1) Isolate DNA and break into fragments (300-800bp) 2) Covalently attach oligonucleotide adapters to 5' and 3' ends of DNA 3) Denature DNA into single strands (mixture is called sample library) 4) Attach single-stranded DNA to beads by adapters (one strand of DNA to bead initially) 5) Emulsify beads in oil-water mixture so one bead per droplet; droplets contain PCR reagents which amplify DNA to ~1million copies per bead 6) Deposit bead in picotiter plate; one bead per well 7) Add sequencing reagents, including primers and allow one nucleotide (in solution) to pass through at a time) 8)Pyrophosphate is released when nucleotide incorporated into growing strand; reacts to produce ATP which reacts with luciferin and in presense of luciferase produces light 9) Synthesis is monitored in real time by a computer (Sequencing by Synthesis) by detecting when each well lights up 10) Use computers to determine one continuous sequence from each well by analyzing overlapping sequences

Early Shotgun Sequencing Process

1) Isolate chromosomal DNA 2) Clone large fragments into BACs or YACs and create a contig for each chromosome 3) For each BAC shear into smaller pieces and clone into vectors 4) Use shotgun sequencing to determine sequence of chromosomal DNA at one end 5) Based on overlapping sequences create one contiguous sequence

Outcome of cytogenic mapping

1) Markers will appear on both homologous chromosomes 2) If sister chromatids are present (depending on cell's place in the cell cycle), two markers will appear on one chromosome (one for each sister chromatid)

Process of Shotgun Sequencing

1) Purify DNA 2) Sonicate DNA to break into fragments (~2000 bp) 3) Clone DNA into vectors 4) Subject many clones to dideoxy DNA sequencing; since this produces many overlapping sequences they can be ordered as they are in the chromosome 5)Use bioinformatics tools to identify types of genes in genome

Cytogenic Mapping Procedure

1) Treat cells with agents that make them swell and adhere to surface of slide 2) Denature chromosomal DNA 3) Add single-stranded DNA probes that have biotin incorporated into them 4) Add fluorescently labeled avidin that binds to biotin 5) View under fluorescent microscope

Why is YAC the best vector for Physical Mapping in humans?

1) Yeast Artifical Chromosome (YAC; up to 2 million bp) while BAC and PAC hold about 300,000 bp 2) A few hundred YAC is enough to create contig of entire chromosome 3) Closely resembles eukaryotic chromosome

Genetic Map

A chart that describes the relative locations of genes and other DNA segments along a chromosome

Contig

A collection of clones containing contiguous, overlapping pieces of chromosomal DNA

Restriction Fragment Length Polymorphisms (RFLPs)

A difference in restriction fragment lengths (difference in the number of palindromes) between different individuals when the same restriction enzyme is applied to both sets of DNA.

Cosmid

A hybrid between a plasmid vector and a phage lambda; hold smaller piece of DNA than YAC, BAC, or PAC

Molecular Marker

A segment of DNA that is found at a specific site along a chromosome and has properties that enable it to be uniquely recognized using molecular tools such as PCR and gel electrophoresis; can be regions of DNA that do not code for genes

Microsatellites

Also known as short tandem repeats (STRs); short repetitive sequences abundantly interspersed through out a species genome; variable in length among different individuals

Limitations of PFGE

Animal and plant chromosomes are generally too large to be resolved through this technique

What does physical mapping require?

Cloning of many pieces of chromosomal DNA

How is a physical map of a chromosome constructed?

Creating a contiguous series of clones that span a chromosome

Three ways to determine the organization of DNA regions

Cytogenic mapping, Linkage mapping, and Physical mapping

RFLP Map

Describes the locations of many different RFLPs through out the genome; can be used to locate functional genes within a genome

How to find the distance between RFLPs

Divide the total number of recombinants by the total number of offspring and multiply by 100 (result in map units)

Why are molecular markers preferable to allelic differences?

Easier to identify molecular markers within a species' genome

Restriction Enzyme

Enzymes that recognize palindromes and can be used to digest a chromosome into many small pieces of different lengths depending on the spacing of the palindromes

Third-generation Sequencing Platforms

High-throughput platforms that read single DNA molecules

Second-generation Sequencing Platforms

High-throughput platforms that uses PCR to amplify DNA

Current uses of PFGE

Identify species of microorganisms or strains of the same species

Uses of PFGE

In physical mapping to 1) purify large pieces of DNA 2) construct a library 3) coarsely map locations of genes 4) In combination with Southern Blotting, cloned genes can be located on a chromosome

Previous uses of PFGE

Isolate large pieces of chromosomes to construct contigs

Which type of mapping uses molecular markers?

Linkage mapping

Cytogenic Mapping

Mapping that relies on the localization of gene sequences within chromosomes that are viewed using a microscope

Physical Mapping

Mapping that uses DNA cloning techniques to determine the location of and distance between genes and other DNA regions in nucleotide base pairs

Linkage Mapping

Mapping that uses the frequency of recombination between different genes to determine their relative spacing and order along a chromosome

Chromosome Walking

Method of ordering fragments of DNA cut by restriction enzymes; Probes prepared by automated nucleotide sequencing of the ends are used to identify overlapping pieces (use more than one restriction enzyme)

Pulsed-field gel electrophoresis (PFGE)

Method used to separate individual chromosomes or large pieces of chromosomes to compare genomes

Genomics

Molecular analysis of the entire genome of a species

PFGE vs. Gel Electrophoresis

PFGE uses alternating pulses of current applied to the gel from different directions

Monomorphic Site

Palindromic site that a particular restriction enzyme digests that is the same for 99% of a population

Use of RFLPs vs. microsatellites

RFLPs use restriction digests and Southern blots while microsatellites use PCR procedures which are generally easier

Results of STS gel for microsatellite sequence in a homozygous individual

Region will be the same length for both copies of the sequence

Results of STS gel for microsatellite sequence in a heterozygous individual

Region will differ in length for the two copies of the sequence

Two types of molecular markers used in linkage mapping

Restriction Fragment Length Polymorphisms (RFLPs) and Microsatellites

Sequence-tagged Site (STS)

Site in DNA that can be amplified among the thousands in a genome through the use of PCR primers

What characterizes chromosome fragments for physical mapping?

Size (by length in bp), the genes they contain, the relative location within a chromosome

How can eukaryotic chromosomes be distinguished?

Size, centromeric location, banding patterns

Sample library

Solution of single-stranded DNA with oligonucleotide adapters; used in pyrosequencing

Sequencing by Synthesis

Technique used in pyrosequencing; synthesis of DNA is monitored in real time

Double-Barrel Shotgun Sequencing

Technique used to sequence chromosomal DNA in a vector from both ends; used in Whole-Genome Shotgun Sequencing

high-throughput sequencing

The ability to rapidly sequence large amounts of DNA; skip cloning steps

Subcloning

The act of taking a small piece of DNA from one vector and inserting it into another vector; needed for chromosome walking

Positional Cloning

The cloning of a gene based upon its mapped position along a chromosome; uses subcloning and chromosome walking

What does the sequence of the entire genome reveal?

The genome size and which genes the organism has

What does functional genomics study?

The interaction of the components of the genome to produce the traits of the organism

What does proteomics study?

The interactions of all cellular proteins to produce the characteristics of particular cell types and the traits of complete organisms

Restriction Fragments

The pieces of the chromosome that result from applying restriction enzymes

locus

The site within a genetic map where a specific gene or other DNA segment is found

How are molecular markers similar to alleles?

They are polymorphic. Within a population they vary from individual to individual allowing the distances between linked markers to be determined from the outcome of crosses.

How can molecular markers be used?

They serve as reference points along a genetic map; determine the approximate location of an unknown gene that causes a human disease

How are palindromes lost or created?

Through mutation.

Goal of physical mapping

To create a map in which the distances between markers are expressed in some physical measure (usually number of nucleotides) rather than recombination frequencies

What is in situ hybridization used for?

To cytogenetically map the locations of genes and other DNA segments within large eukaryotic chromosomes, while chromosomes are adhered to a surface or held in place

What is the goal of cytogenic mapping?

To determine the location of genes along an intact chromosome; locate genes relative to the banding pattern of a chromosome

Goal of Linkage Studies

To follow the transmission of many different microsatellites to determine which are linked along the same chromosome and which are not; with a large pedigree, crossing over can be identified and map distances can be found

Genome

Total genetic composition of an organism

How to obtain a discrete banding pattern for chromosomes (cytogenic mapping)?

Treat chromosomes with dyes

Fluorescent in situ hybridization (FISH)

Type of in situ hybridization where the probe is fluorescently labeled.

Vectors for clones in Physical Mapping

YAC, BAC, and PAC (allows large amounts of DNA to be cloned)

Members of contigs may contain:

genes, RFLPs, or STSs.

Chromosome Mapping

the experimental process of determining the relative locations of genes or other segments of DNA along individual chromosomes

How often is a microsatellite found?

~ every 10,000 bases