1. What is Haplotyping? T C A G
Diploid organisms may be polyallelic at some loci (highlighted to the left) A C C G
SNP sequencing would show the genotype as A/T, C, A/C, G
This would still leave two possibilities for the true haplotype: ACCG/TCAG or ACAG/TCCG.
Single-molecule haplotyping can distinguish between chromosomes, and tell us that the true haplotype is ACCG/TCAG
Haplotypes give more information, and have better predictive power than SNP sequences

2. Three Underlying Technologies
ACCTGTCAGGCGTACCA
TGGACAGTCCGCATGGT
Padlock probe labeling is used to allele-specifically label the SNPs of interest.
Molecular combing is used to stretch the DNA molecules on a surface prior to imaging
FIONA is used to localize the labels.
Combining all three gives barcoded DNA

3. Molecular Haplotyping
Why?
Diploid organisms (e.g. humans) may be heterozygous
Knowing the correlations of SNPs on each individual chromosome (the haplotype) confers more predictive power than SNPs alone
Most genotyping techniques use bulk PCR products, and cannot distinguish chromosomes
Single molecule analysis can allow us to distinguish discrete populations
How?
First, DNA is allele specifically labeled with single fluorescent probes using padlock-probe labeling
Second, DNA is stretched onto a surface using molecular combing
DNA backbone and fluorescent nucleotide labels are imaged using FIONA
Double stranded DNA can be stretched with molecular combing onto a surface, as shown. Single stranded DNA can potentially be stretched if it is stabilized with RecA, a single-stranded DNA binding protein. Labeling of ssDNA with padlock probes can be more efficient with ssDNA.

4. Haplotyping and Genomics
Current genotyping technologies work on bulk PCR products, and hence cannot distinguish heterozygous haplotypes, because they cannot distinguish products from different chromosomes
Haplotypes have much more predictive power than simple SNP sequencing for determining disease susceptibility and drug interactions
Once general patterns have been discerned using genome-wide SNP scans, haplotyping will be the dominant means of determining precise correlations between regions of genomic interest and disease.
We will focus on a 500 kb region containing the HOXA locus on human chromosome 7, as part of the ongoing work of the International HapMap consortium.

5. Single Molecule Fluorescence Haplotyping
3 kbp
17 kbp
In our approach, PCR products are site- and allele-specifically labeled with single dye molecules, and are imaged to establish a “barcode” which can be used to determine the haplotype at selected SNPs.
Different alleles are labeled with different dyes (e.g. Cy3, Cy5), and can be distinguished by color