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| Hello, Dolly! |
1997—Dolly became the first of many cloned animals resulting from somatic cell nuclear transfer |
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mcs5109 Sat, 31 Oct 2009 05:04:42 GMT |
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| Ribozymes |
-discovered in 1981
-provided novel way of preventing unwanted gene expression (ex:
cancer and AIDS) |
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mcs5109 Sat, 31 Oct 2009 05:03:15 GMT |
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| Altman and Cech |
awarded Nobel Prize for their
discovery of ribozymes |
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mcs5109 Sat, 31 Oct 2009 05:03:15 GMT |
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| first recombinant vaccine |
to Hepatitis B
was developed in 1987 |
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mcs5109 Sat, 31 Oct 2009 05:03:15 GMT |
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| Human Gene Therapy |
-approved in 1990
-ADA deficiency treated with lymphocytes engineered with
retroviral ADA gene
-responses were positive but
efficacy could never be proven
-SCID X1 treatment was effective, but resulted in 2 children developing leukemia |
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mcs5109 Sat, 31 Oct 2009 05:03:15 GMT |
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| HGP fun facts |
-Human genome comprised of only 30,000 genes
-Proteome may explain difference in complexity of organisms
-Most common type of DNA sequence is SNP
-Hundreds of human genes originated from horizontal
transfer from bacteria or have evolved from transposable elements |
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mcs5109 Sat, 31 Oct 2009 04:56:41 GMT |
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| Two different strategies HGP strategies |
Celera Company (Craig Venter)
-Used shot‐gun approach
-blasted the genome into small fragments, each sequenced individually, then used computer software to align
-faster, but less accurate |
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mcs5109 Sat, 31 Oct 2009 04:53:40 GMT |
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| Two different strategies HGP strategies |
-DOE/NIH (Francis Collins)—many labs throughout the world carefully sequenced each base in the
segment of DNA they were allotted
-Parallel projects were completing sequences of smaller genomes |
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| Initial goals of the HGP |
-Mapping & sequencing human/model organisms genome
-ID 30,000 genes
-Software & database development
-transfer technology to private sector |
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| Human Genome Project |
-Began as government funded joint project by DOE (for nuclear weapons) and NIH
-not just “human” DNA |
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mcs5109 Sat, 31 Oct 2009 04:47:58 GMT |
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| making human genome map... |
-variability of polymorphisms led Jeffreys et al to propose a method of DNA fingerprinting
-produce unique profiles to ID humans
-SNPs were proposed as means to produce genome map |
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mcs5109 Sat, 31 Oct 2009 04:47:58 GMT |
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| Mutation Detection |
-Direct detection through sequencing
-preferred method
-expensive!
-Other methods to detect mutations: DNA scanning, linkage analysis |
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mcs5109 Sat, 31 Oct 2009 04:47:58 GMT |
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| SNP |
-Single Nucleotide Polymorphism
-Potentially the most useful
-difficult to detect because they may not alter the recognition
sequence, or the length of the restriction fragment
-used in gene discovery research to
ID small differences which may result in
significant phenotypic variabilities
-$$$ |
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mcs5109 Sat, 31 Oct 2009 04:40:35 GMT |
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| SSR |
Simple Sequence Repeats (also called microsatellites)
-much smaller
-can only be detected by PCR
-small repeats make fragments different lengths |
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mcs5109 Sat, 31 Oct 2009 04:38:30 GMT |
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| VNTR |
-Variable Number of Tandem
-potentially more informative, as there are more possible variants
-the restriction fragments would vary
in size based on the difference in the number of repeats |
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mcs5109 Sat, 31 Oct 2009 04:37:16 GMT |
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| RFLP |
-Restriction Fragment Length Polymorphism
-segment of DNA is digested by restriction enzymes
-the resulting fragments will be varying
lengths, due to the differences in recognition sites |
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mcs5109 Sat, 31 Oct 2009 04:35:56 GMT |
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| Polymorphisms |
-Most changes in DNA don't lead to disease
-the majority do not effect function, unless it falls in regulatory region (controls expression)
-RFLP
-VNTR
-SSR
-SNP |
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mcs5109 Sat, 31 Oct 2009 04:33:54 GMT |
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| DNA Mutation Terminology |
-Δ means deletion; ex: ΔF508 is a deletion of the amino acid phenylalanine at position 508
-Cys282Tyr means there is a cysteine to tyrosine substitution at position 282
-use single letters instead |
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mcs5109 Sat, 31 Oct 2009 04:33:54 GMT |
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| Clinical phenotypes of chromosomal abnormalities |
-Developmental delay/mental retardation
-facial morphogenesis
-Growth delay
-Congenital malformations, especially heart defects |
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mcs5109 Sat, 31 Oct 2009 04:28:28 GMT |
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| Ring chromosome |
material is deleted at both ends, the new ends join to form a ring |
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mcs5109 Sat, 31 Oct 2009 04:25:32 GMT |
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| Duplication |
section of chromosome is duplicated
(extra genetic material) |
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| Inversion |
section of chromosome is snipped out and reinserted upside down |
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| Translocation |
section of chromosome attached to another chromosome |
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| Microdeletion |
minute amount of material is missing |
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mcs5109 Sat, 31 Oct 2009 04:25:32 GMT |
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| Deletion |
small section is missing |
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| Chromosomal Structure Abnormalities |
Deletion
Microdeletion
Translocation
Inversion
Duplication
Ring chromosome |
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mcs5109 Sat, 31 Oct 2009 04:25:32 GMT |
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| Klinefelter |
2 or more X chromosomes w/Y |
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mcs5109 Sat, 31 Oct 2009 04:21:55 GMT |
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| Triple X |
extra X chromosome |
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mcs5109 Sat, 31 Oct 2009 04:21:55 GMT |
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| Turner Syndrome |
missing all of part of one X |
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mcs5109 Sat, 31 Oct 2009 04:21:55 GMT |
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| Edwards Syndrome |
Trisomy 18 |
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| Patau Syndrome |
Trisomy 13 |
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| Down Syndrome |
Trisomy 21 |
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mcs5109 Sat, 31 Oct 2009 04:21:55 GMT |
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| Abnormality of Number |
Abnormal meiosis‐ cells divide abnormally, resulting in sperm or egg cells with too many or too few
chromosomes
Sister chromatids don’t separate properly → resulting
cells abnormal
One cell has too many chromosomes → trisomy
One cell has too few chromosomes → monosomy |
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mcs5109 Sat, 31 Oct 2009 04:21:55 GMT |
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| Chromosomal Abnormalities |
Abnormalilities of number
-Polyploidy
-Aneuploidy
Abnormalities of structure
-Deletions
-Microdeletions
-Translocations
-Inversions
-Duplication
-Ring chromosome |
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mcs5109 Sat, 31 Oct 2009 04:16:40 GMT |
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| SKY |
Spectral Karyotyping |
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mcs5109 Sat, 31 Oct 2009 04:15:03 GMT |
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| FISH |
-Fluorescence in situ hybridization
-greater resolution of chromosomes
-fluorescently labelled probes used to identify specific loci on chromosomes
Useful in identifying chromosomal abnormalities:
numerical (aneuploidies & polyploidies), structural
(translocations, deletions) & mixtures (mosaicism) |
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mcs5109 Sat, 31 Oct 2009 04:15:03 GMT |
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| chromosome regions |
-chromosomes (p and q) are divided into regions
-IDs specific genes locations
EX: 7q31.3 = chromosome 7, long
arm, region 3, band 1, sub‐band .3 |
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mcs5109 Sat, 31 Oct 2009 04:15:03 GMT |
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| G‐banding |
-banding pattern from giemsa staining
-IDs each chromosome |
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mcs5109 Sat, 31 Oct 2009 04:15:03 GMT |
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| Karyotype |
the chromosomal makeup (number, size
and morphology) in a cell, individual or species |
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mcs5109 Sat, 31 Oct 2009 04:08:48 GMT |
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| Cytogenetics |
the study of chromosomes |
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mcs5109 Sat, 31 Oct 2009 04:08:48 GMT |
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| giemsa staining banding pattern |
-euchromatin stains lighter
-heterochromatin stains darker |
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mcs5109 Sat, 31 Oct 2009 04:08:48 GMT |
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| chromosome structure |
Double Helix →
Nucleosomes →
Chromatin →
Chromatids |
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mcs5109 Sat, 31 Oct 2009 04:06:18 GMT |
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| telomere |
tandem repeats |
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mcs5109 Sat, 31 Oct 2009 04:06:18 GMT |
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| Centromere |
-divides chromosomes into short (p for petite) and long (q for not p) arms |
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mcs5109 Sat, 31 Oct 2009 04:06:18 GMT |
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| PCR Problems |
-very sensitive
-minute amounts of contamination can be amplified
-fidelity of polymerases‐ error rate is ~ 1 nucleotide/400 bases
-old/decaying samples
-LCN PCR can lose sequence
specificity |
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mcs5109 Sat, 31 Oct 2009 04:06:18 GMT |
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| Q‐PCR |
-Quantitative PCR
-allows products to be measured in real time |
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mcs5109 Sat, 31 Oct 2009 03:55:56 GMT |
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| In Situ PCR |
‐allows genes or mRNA to be amplified from tissues (used to identify tissue‐specific genes) |
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mcs5109 Sat, 31 Oct 2009 03:55:56 GMT |
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| Multiplex PCR |
‐uses several sets of primers in one
reaction (this is used on forensic DNA analysis) |
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mcs5109 Sat, 31 Oct 2009 03:55:56 GMT |
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| RT‐PCR |
-Reverse Transcriptase PCR
-uses RNA as a starting product |
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mcs5109 Sat, 31 Oct 2009 03:55:56 GMT |
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| copies made by PCR |
2^n |
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mcs5109 Sat, 31 Oct 2009 03:54:11 GMT |
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| Taq polymerase |
-heat resistant |
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mcs5109 Sat, 31 Oct 2009 03:54:11 GMT |
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| PCR Steps and Temps |
Denaturation‐ 94
Annealing‐ 59
Elongation‐ 72 |
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mcs5109 Sat, 31 Oct 2009 03:54:11 GMT |
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| When/who developed PCR? |
developed in 1985 by Mullis, Saiki and colleagues at Cetus Corporation |
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mcs5109 Sat, 31 Oct 2009 03:54:11 GMT |
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