1. Copyright © 2010 Pearson Education Inc.
Lecture 23 DNA Polymorphisms Based on Chapter 10 Recombinant DNA Technology
Copyright © 2010 Pearson Education Inc.

2. 1. Questions you should be able to answer from today’s lecture.
What types of DNA polymorphisms are present in the genome?
How can DNA polymorphisms be used in genetic analysis and in disease diagnosis?
What is DNA fingerprinting (DNA typing) and how can it be used?
How does gene therapy work?
How are the techniques used to clone, amplify, and manipulate DNA applied commercially in the biotechnology industry?
How can plants be engineered genetically?

3. 2. Uses of DNA Polymorphisms in Genetic Analysis
Genes have historically been used as markers for genetic mapping experiments.
A DNA polymorphism is two or more alleles at a locus that vary in nucleotide sequence or number of repeated nucleotide units (indels).
DNA markers are polymorphisms suitable for mapping, used in association with gene markers for genetic and physical mapping of chromosomes.
Slide 2 - Uses of DNA Polymorphisms in Genetic Analysis
7. Genes have historically been used as markers for genetic mapping experiments.
8. A DNA polymorphism is two or more alleles at a locus that vary in nucleotide sequence or number of repeated nucleotide units (indels).
9. DNA markers are polymorphisms suitable for mapping, used in association with gene markers for genetic and physical mapping of chromosomes.
9.1. DNA markers are distinguishable polymorphic alleles that do not encode proteins, and therefore are neither dominant nor recessive.
9.2. Map distances may be calculated using a combination of genes and DNA markers, for greater resolution.
10. Classes of DNA Polymorphisms
There are three major classes of DNA polymorphisms:
Single nucleotide polymorphisms (SNPs).
Short tandem repeats (STRs).
Variable number tandem repeats (VNTRs).

4. 3. Single Nucleotide Polymorphism (SNPs, “Snips”): Southern Blot
Slide 3 - Single Nucleotide Polymorphism (SNPs, “Snips”)
5. Single nucleotide polymorphisms (SNPs, “snips”) are base-pair differences between individuals, as explained in Chapter 8.
5.1. A few SNPs create or abolish restriction sites, resulting in restriction fragment length polymorphisms (RFLPs). RFLPs are detected by (Figure 10.14):
Southern hybridization:
Isolates genomic DNA and digests with a restriction enzyme.
Electrophoreses and transfers DNA to a membrane filter.
Probes with labeled DNA from the polymorphism region.
Monozygotes show one band, heterozygotes two.

5. 4. Single Nucleotide Polymorphism (SNPs, “Snips”): PCR
Slide 4 – Single Nucleotide Polymorphism (SNPs “Snips”): PCR
PCR amplification (Figure 10.15):
Isolates genomic DNA and amplifies sequence of interest with specific primers.
Digests amplified DNA with appropriate restriction enzyme.
Analyzes fragments produced with agarose gel electrophoresis.

6. 5. Detection of All SNPs Slide 5 - Detection of All SNPs
6. Most DNPs do not alter restriction sites, so other methods are used for analysis:
6.1. Allele-specific oligonucleotide (ASO) hybridization is used for single SNPs (Figure 10.16).
Oligonucleotide complementary to one SNP allele is mixed with target DNA and hybridized at high stringency.
If hybridization occurs, target DNA has the allele corresponding to the oligo.
Hybridization does not occur if a different allele is present.

7. 6. Short Tandem Repeats (STRs) & Variable Number Tandem Repeats (VNTRs)
Slide 6 - Short Tandem Repeats (STRs) & Variable Number Tandem Repeats (VNTRs)
7. Short tandem repeats (STRs), or microsatellite sequences, contain very short (2–6-bp) tandem repeats and are highly polymorphic.
7.1. Examples are the dinucleotide repeat (GT)n and the trinucleotide repeat (CAG)n.
7.2. STRs are distributed widely in the human genome, with thousands of sites currently known.
7.3. Many are polymorphic and are used for genetic mapping and forensics.
7.4. STRs are usually typed by PCR with primers flanking the sequence (Figure 10.17).
A population may have many different allele lengths for STRs.
An individual may be either homozygous or heterozygous for aparticular STR.
Variable Number Tandem Repeats (VNTRs)
8. Variable number tandem repeats (VNTRs), also called minisatellites, are longer than STRs (7 or more bp).
8.1. There are far fewer VNTR loci in the human genome than STR loci.
8.2. To detect VNTR polymorphisms, PCR is not generally useful, and instead, restriction digestion and Southern blotting are used.
DNA is digested with a restriction enzyme that cuts flanking the VNTR.
Fragments are electrophoresed and then blotted to a filter.
The blot is probed with the VNTR repeating sequence.
(1) Some VNTR sequences are in only one genomic locus, corresponding to a monolocus probe.
(2) Other VNTR sequences map to a number of genomic loci, corresponding to a multilocus probe.

8. 7. DNA Molecular Testing for Human Genetic Disease Mutations
DNA testing is increasingly available for genetic diseases, including:
Huntington disease.
Hemophilia.
Cystic fibrosis.
Tay–Sachs disease.
Sickle-cell anemia.
Slide 7 - DNA Molecular Testing for Human Genetic Disease Mutations
 9. Many human diseases result from protein defects caused by DNA mutations. DNA testing is increasingly available for genetic diseases, including:
9.1. Huntington disease.
9.2. Hemophilia.
9.3. Cystic fibrosis.
9.4. Tay–Sachs disease.
9.5. Sickle-cell anemia.
10. Designing DNA molecular tests requires knowledge of gene mutations that cause a disease, derived from sequencing the gene involved.
 11. Often a disease is caused by a variety of mutations, complicating its study. The breast cancer genes BRCA1 and BRCA2 are examples:
Normal BRCA1 and BRCA2 genes control cell growth in breast and ovarian tissue.
Mutations in the BRCA1 and BRCA2 genes can lead to cancer. Hundreds of mutations in these genes have been identified.
Each BRCA1 or BRCA2 mutation confers a different risk of developing cancer, ruling out a single DNA molecular test to assess an individual’s breast cancer risk associated with these genes.
 12. Genetic testing reveals the presence of a mutation associated with a genetic disease. Genetic testing is usually done on a targeted population of people with symptoms or a family history of the disease.
 13. Genetic testing is distinct from genetic screening, which involves the population at large, and it is also distinct from diagnostic testing to determine whether a disease is present, or the extent of its development.

9. 8. Purposes of Human Genetic Testing
Human genetic testing serves three main purposes:
Prenatal diagnosis - uses amniocentesis or chorionic villus sampling to assess risk to the fetus of a genetic disorder
Newborn screening .- examples of tests for specific mutations using blood from newborns include:
Phenylketonuria (PKU).
Sickle-cell anemia.
Tay–Sachs disease.
Carrier (heterozygote) detection - Carrier testing is now available for many genetic diseases, including:
Huntington disease.
Duchenne muscular dystrophy.
Cystic fibrosis.
Slide 8 - Purposes of Human Genetic Testing
14. Human genetic testing serves three main purposes:
Prenatal diagnosis.
Newborn screening.
Carrier (heterozygote) detection.
 15. Prenatal diagnosis uses amniocentesis or chorionic villus sampling to assess risk to the fetus of a genetic disorder by analyzing for a specific mutation, or biochemical or chromosomal abnormalities.
If both parents are carriers (heterozygotes) for the mutant allele, the probability is 1⁄4 that the fetus is an affected homozygote, 1⁄2 that it is a carrier, and 1⁄4 that it is homozygous for the normal allele. Genetic testing can determine the result of a particular conception.
Genetic testing may be used during in vitro fertilization to eliminate before implantation embryos with mutated genes that could result in serious disease.
16. Carrier testing is now available for many genetic diseases, including:
Huntington disease.
Duchenne muscular dystrophy.
Cystic fibrosis.
17. Examples of tests for specific mutations using blood from newborns include:
Phenylketonuria (PKU).
Sickle-cell anemia.
Tay–Sachs disease.

10. 9. Examples of DNA Molecular Testing: Testing by Restriction Fragment Length Polymorphism
Slide 9 - Examples of DNA Molecular Testing: Testing by Restriction Fragment Length Polymorphism
18. Testing by restriction fragment length polymorphism (RFLP) analysis detects loss or addition of a restriction site in the region of a gene.
The restriction map is independent of gene function, so RFLPs may occur without changing the phenotype.
RFLP marker information is used in the same way as “conventional” DNA markers and is assayed directly in the form of a restriction map.
In heterozygotes both parental types are seen, allowing easy detection of carriers.
19. RFLPs are associated with many genetic disorders. Sickle-cell anemia is an example:
A single base-pair change in the b-globin gene results in abnormal hemoglobin, Hb-S, rather than the normal Hb-A. Hb-S molecules cause sickling of red blood cells.
The Hb-S mutation is an AT-to-TA base-pair change in the sixth codon of b-globin, resulting in a valine rather than a glutamic acid and eliminating a DdeI restriction enzyme site (Figure 10.18).

11. Slide 10 – Example Sickle Cell Disease Genetic Testing
In the normal b-globin (Hb-A) gene there are three DdeI sites, while the sickling form, Hb-S, has only two DdeI sites. This difference can be detected using Southern hybridization of genomic DNA with a b-globin gene probe (Figure 10.19).
20. RFLPs associated with genetic disorders may also result from changes in flanking sequences. PKU is an example:
PKU results from defective phenylalanine hydroxylase enzyme.
Genomic DNA digested with HpaI, Southern blotted, and probed with cDNA probe from phenylalanine hydroxylase mRNA shows different restriction fragments for PKU and normal individuals.
The RFLP results from DNA sequences located 39 to the gene that usually segregate with it. Recombination events that occur between the site of the RFPl and the gene mutation can complicate this test.

12. 11. Examples of DNA Molecular Testing: Testing Using PCR Approaches
Slide 11 - Examples of DNA Molecular Testing: Testing Using PCR Approaches
21. PCR is another approach to DNA molecular testing. It requires sequence information so that specific primers can be designed. An example is allele-specific oligonucleotide (ASO) hybridization used to detect mutations in GLCIA, a gene involved in maintaining normal eye pressure (Figure 10.20).
Abnormal pressure in the eye results in glaucoma, which can cause blindness. Mutations in GLC1A can be responsible for this condition.
Sequence of GLC1A is known and glaucoma-inducing mutations identified.
PCR primers were designed to amplify a region of the gene where glaucoma-inducing mutations occur.
PCR products undergo agarose gel electrophoresis, are extracted from the gel and dotted onto duplicate membrane filters, and are denatured to single strands.
One blot is probed with a labeled oligonucleotide specific to the wild-type allele, while the other receives labeled oligo specific for a particular mutation.
(1) Signal only with the wild-type oligo indicates an individual homozogous for the normal allele.
(2) Signal only with the mutant oligo shows an individual homozygous for the mutant GLC1A allele and at risk of developing glaucoma.
(3) Signal on both filters indicates a heterozygous (carrier) individual.
22. A related PCR procedure uses labeled PCR product as a probe against a filter blotted with an array of normal and mutant alleles of the gene. The dot(s) to which the probe binds indicate the genotype of the individual.

13. 12. Examples of DNA Molecular Testing: DNA Microarrays in Disease Diagnosis
Results given as ratios
Images use colors:
Cy3 = Green
Cy5 = red
Yellow
Yellow is equal intensity or no change in expression
Slide 12 - Examples of DNA Molecular Testing: DNA Microarrays in Disease Diagnosis
23. DNA microarrays are used in screening for genetic diseases. An example is mutations in the BRCA1 and BRCA2 genes, important in hereditary breast and ovarian cancers.
Hundreds of different mutations in these genes are known, and so microarrays are used to detect them.
Genomic DNA of the patient is compared with that from a normal individual.
The patient’s DNA is used as a PCR template for the BRCA1 and BRCA2 genes, which are labeled with Cy3 (green).
Normal DNA from these regions is labeled with Cy5 (green) and mixed with the patient’s PCR amplicons on the DNA microarray.
The microarray is small probes covering the entire BRCA1 and BRCA2 genes.
Normal DNA hybridizes with all probes. If the patient’s DNA also hybridizes, the spot will appear yellow. A mutation in the patient’s DNA that prevents hybridization results in a red spot, localizing the mutation to a specific site within the affected gene.

14. 13. DNA Typing Slide 13 - DNA Typing
24. DNA typing (DNA fingerprinting or DNA profiling) identifies DNA from particular individuals, using techniques similar to those described for DNA molecular testing for genetic diseases.
25. DNA Typing in a Paternity Case
DNA typing in a paternity case would proceed as follows (Figure 10.21):
DNA samples (typically from blood) are obtained from mother, baby, and putative father.
DNA is cut with a particular restriction enzyme, electrophoresed, and transferred to a membrane filter by Southern blotting.
Hybridization is performed with a labeled monolocus STR or VNTR probe, and the banding pattern is analyzed.
Baby and mother are expected to share one allele, while baby and father share the other allele.
If the man and baby do not share a common allele, DNA typing has proved he is not the father. If they do share an allele, paternity is possible but not proven, since other men also carry the allele at some frequency that can be calculated.
Often five or more different polymorphisms are characterized. If all match with the putative father, the combined probabilities calculated for the array of polymorphisms can be convincing evidence in court.
  DNA typing is not generally accepted for proving parenthood or guilt, although it is widely used as evidence of innocence. DNA evidence is most commonly rejected for procedural reasons, such as errors in evidence collection or processing, or due to lack of population statistics for the alleles in question.
  PCR is the common commercial method for paternity testing.

15. 14. Applications of DNA Typing
Examples of DNA typing used to analyze samples include:
Crime scene invetigation
Population studies to determine variability in groups of people.
Proving horse pedigrees for registration purposes.
Conservation biology to determine genetic variation in endangered species.
Forensic analysis in wildlife crimes, allowing body parts of poached animals to be used as evidence.
Detection of pathogenic E. coli strains in foods
Detection of genetically modified organisms (GMOs) in bulk or processed foods
Slide 14 - Applications of DNA Typing
26. Examples of DNA typing used to analyze samples include:
Crime scene invetigation
Population studies to determine variability in groups of people.
Proving horse pedigrees for registration purposes.
Conservation biology to determine genetic variation in endangered species.
Forensic analysis in wildlife crimes, allowing body parts of poached animals to be matched and used as evidence.
Detection of pathogenic E. coli strains in foods such as ground meat.
Detection of genetically modified organisms (GMOs) in bulk or processed foods, by the presence of inserted sequences.
DNA typing is increasingly being used on much older samples, as well.
DNA extracted from the remains of ancient organisms can be analyzed. Examples include an insect in amber (40 million years old), a fossil leaf (17 million years), and a mammoth (40,000 years).
Historic questions can be addressed by DNA typing. An example is the lingering doubt about whether a boy who perished in captivity in 1795 was the sole surviving son of Louis XVI and Marie Antoinette. Comparison of DNA from preserved tissue and hair, and from living descendants, shows that the dead boy was indeed the dauphin, the heir to the French throne.