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Sickle-cell anemia Caused by single bp substitution in -globin gene
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Sickle-cell anemia

Jan 23, 2016

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Sickle-cell anemia. Caused by single bp substitution in b -globin gene. Determine b -globin genotype by RFLP analysis. Other methods to diagnose genotypes. Allele-specific Oligonucleotides (ASOs): Make two oligonucleotide probes: (a) Normal sequence (b) Sequence of mutant allele - PowerPoint PPT Presentation
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Page 1: Sickle-cell anemia

Sickle-cell anemia

Caused by single bp substitution in -globin gene

Page 2: Sickle-cell anemia

Determine -globin genotype by RFLP analysis

Page 3: Sickle-cell anemia

Other methods to diagnose genotypesAllele-specific Oligonucleotides (ASOs):

Make two oligonucleotide probes:(a) Normal sequence(b) Sequence of mutant allele

Hybridize each oligo (separately) to Southern blot of DNA.Use conditions that allow only oligonucleotides that

are 100% complementary to DNA on blot to hybridize.If only normal oligo hybridizes---homozygous normal alleleIf only mutant oligo hybridizes --- homozygous mutant allele

If both oligos hybridize --- heterozygous

Screen DNA by PCR:Similar to ASOs, in combination with a 2nd primer, use:

(a) PCR primer with normal sequence(b) PCR primer with mutant sequence

Will only get PCR product if the primer perfectly matches the genomic DNA.Amplify gene region by PCR & sequence it.

Page 4: Sickle-cell anemia

Most of the mutations responsible for other genetic diseases do not change a restriction site (i.e., not detectable as RFLP or SSLP)

Multiple mutant alleles for many of these genes.Therefore, unless you know the specific allele an individual might carry, would need to do multiple tests with different allele-specific oligos. Or -- Can detect ~all mutant alleles by sequencing.

Page 5: Sickle-cell anemia

Genome Projects

1. Determine DNA sequence of entire genome.

2. Map the locations and structures of all genes (annotation).

Page 6: Sickle-cell anemia

Two Alternative Approaches

- Ordered clone sequencing:Assemble physical map = set of overlapping clones.Choose smallest number of clones needed to cover entire genome.Sequence just these clones.

- Whole genome shotgun sequencing:Randomly select many clones.Determine their sequences.Use computers to align sequences.

1. Determine DNA sequence of entire genome.

Page 7: Sickle-cell anemia

Ordered Clone Sequencing

Assemble Physical Map:

1. Select random BAC clones and sequence ends of inserts.Each of these sequences = Sequence-Tagged Site (STS).

2. Design PCR primers from within these STS’s & test other BAC clones for inclusion of this DNA sequence by PCR.

3. Assemble clones into “contigs”.Contigs = set of overlapping clones.

Page 8: Sickle-cell anemia

Ordered Clone Sequencing

Contigs

Page 9: Sickle-cell anemia

Whole genome shotgun sequencing

Random clones selected and sequenced - each insert only a few kb.

Can use STS’s from ordered clones to help fill gaps in sequence.

STS#1 STS#2

BAC clone

Page 10: Sickle-cell anemia

Virtual Cloning

Do computer searches of genome databases to identify genes with similar sequences