Other forms of cloning and analysis

1. Other forms of cloning and analysis • PCR • Restriction mapping • The human genome project • In situ methodologies • These adapt southern, northern and western techniques to assess genes and gene expression in cells directly • Sister Chromatid Exchange • An example from start to finish • Cloning the gene responsible for alkaptonuria

2. What PCR can do • It can specifically amplify any DNA sequence from out of a background of contaminating sequence • The only specific reagents necessary are primers that define the amplified region • The PCR product will geometrically increase in quantity as a sequence between these two primers • PCR is conceptually a method of chemically cloning a DNA sequence

3. PCR advantages over cloning • Faster • Days to weeks to rescue a DNA sequence using standard techniques • PCR takes a day from design to completion • The actual reaction takes a few hours • Cheaper • In time and materials • More versatile • The polymerase chain reaction can be adapted to fuse proteins, and create deletions, insertions and substitution mutations with the same economy and speed as using PCR for cloning purposes

4. PCR disadvantages • Errors • This used to be more of a problem than it currently is, however the thermostable polymerase Taq has no editing function, and therefore cannot correct errors of incorporation • In addition, the reaction is carried out at 72C and is raised to 98C or higher. This increases oxidative damage to the DNA • Size • PCR products rarely get over 10 kb, and are usually only a few thousand bases long • PCR products are difficult to clone • PCR products are often used as inserts for plasmid clones because of their ease of preparation, BUT • Taq polymerase adds an A onto the 3’ end of its product

5. Restriction mapping • The strategy may be employed to map an entire genome • Clones contain multiple restriction sites • The arrangement of the restriction sites may be determined by cutting DNA with single and multiple enzymes followed by size assessment on an agarose gel • Enzyme 1 cuts the linear DNA twice and enzyme 2 cuts once • The relative arrangement of the restriction sites is logically given by the change in sizes of the fragments following a double digest • 3 kb becomes 1+2 • 10 kb becomes 8+2 • 7 kb becomes 6+1

6. The human genome project • This was an intensive effort to clone and sequence the entire human genome • Once sequenced the data was to be analyzed and turned over to the public • It was carried out by a national consortium of federal and University laboratories • It became a competition with a private company: Celera • The “race” was essentially a dead heat, with the private company claiming patent rights over the sequences they obtained first and the consortium turning its data over to the public domain • With everything cloned and sequenced, it is now possible to “clone by phone” any gene of piece of human DNA if some sequence data is acquired or if the chromosomal location of the gene is known

7. The approaches to cloning the human genome Human genomic libraries were made and either • Systematically organized and assembled into a map • "Shotgun" sequenced

8. The NIH approach • Laboratories were assigned specific sequences such as chromosomes to work on • Chromosome specific libraries were generated • The library was mapped using restriction enzymes • Each clone within the library was oriented with relation to the other clones • This created an entire map of the chromosome • The clones were sequenced

9. The Celera approach • A human genomic library was used as a source of clones for sequencing purposes and the clones sequenced without regard for a map • The overall sequence of the genome was assembled because of overlap between the clones

10. In situ hybridization techniques • The general location of a gene can be determined in humans often using standard genetics • We will talk about genetic mapping later • To determine the exact location of a gene on a chromosome, cloned DNA may be hybridized to a metaphase chromosome squash directly • The DNA probes are fluorescence labeled and are very large (50 kb) • In-situ hybridization both locates the gene and can assess problems with the gene • Below Chromosome 7 is missing DNA near the centromere in Williams syndrome

11. Microdeletion Syndromes

12. Sister chromatid exchange (SCE) • Homologous recombination takes place during mitosis as well as meiosis • But homologous chromosomes do not line up • Instead the sister chromatids exchange arms • Since the arms are identical it has in theory no genetic effect • However SCE increases under conditions of increased mutagenesis • Exposure to mutagens • Predisposing genetic conditions • Since it can be detected it serves as a marker for those conditions

13. Detecting SCE • In culture cells are grown in the presence of bromodeoxyuridine (the thymidine analog) for two rounds of cell division • Cells are then used to make metaphase chromosomes • One round of replication produces two sister chromatids that contain half BUdR and half thymidine • The second round of replication produces one sister chromatid that contain half BUdR and half thymidine, and another sister chromatid that contains only BUdR.

14. Normal Detecting SCE II • Cells after the second round of division will therefore have only one of the four DNA strands of a metaphase chromosome labeled with thymidine • Metaphase spreads are made and stained with a dye that stains thymidine containing strands more than BudR containing strands • SCE is visible by the exchange of arms in the metaphase chromosomes High frequency SCE

15. Genbank • This is a national computerized database containing sequence from any source including • mRNA • Genomic clones • Engineered DNA (often patented) • It includes relevant data as to who has the clone (so you can get some – it’s free!) • If you have a sequence, you can search Genbank and determine if your sequence matches anything that is known • With the completion of the human genome project, all known human sequence is in the database • The sequence information permits the design of PCR primers and therefore amplification of any sequence in the human genome

16. Homogentisic acid 1,2 dioxygenase (Or HGO to the cognoscenti) • In 1902 Archibald Garrod described the disease alkaptonuria as a buildup of homogentisic acid • Alkaptonuria is first recognized when urine turns black on standing • There’s Archie • The pathway for breakdown of homogentisic acid was determined, and 10 years later Garrod also proposed that the defect was in the enzyme that converted homogentisic acid to maleylaectoacetic acid • That would be HGO • He was right

17. Fast forward 84 years to 1992 • Using genetic techniques, the gene for HGO was mapped in humans to chromosome 3 • Specifically 3q2, or the second band on the long arm of the chromosome • 1995, a gene for HGO was cloned from the yeast • The sequence was used to search genbank and a human clone was discovered that was 52% similar to the yeast gene

18. Confirmation of a successful clone-by-phone • The cataloged candidate HGO cDNA clone was recloned into an expression vector • Knowing the sequence permitted alignment of the open reading frame with the Shine-Delgarno sequence of the expression vector • This candidate HGO clone made protein in bacteria with HGO activity • A northern blot using the cloned DNA as probe and liver RNA as target resulted in a single band reflecting the HGO mRNA • HGO is a liver enzyme • These data indicate the DNA represents the HGO gene

19. Further studies • The cDNA was used as probe to rescue a genomic clone • In situ hybridization using the clone paints chromosome 3 at band q2 • DNA taken from people with alkaptonuria was amplified using HGO specific primers and PCR • In a few hours, enough DNA was available for sequencing • HGO mutations responsible were identified and could be tracked in families • The probability of transmission can be determined in couples that are having kids • The genetic constitution of a fetus in utero can also be addressed

20. An ethical dilemma • Alkaptonuria is not a lethal defect • Arthritis due to the buildup of black pigment in the joints • It sometimes makes its appearance late in life • It is treatable with high doses of vitamin C • This decreases the affinity of the metabolite for cartilage and may affect the arthritis • And other drugs • But if you knew early in term that a fetus was going to have no functioning HGO, what would you do? • As well, if you could stop the birth of the four sisters with androgen insensitivity? • Science gives you, the physician, the power of prediction, but does not tell you how or when to use it