UNIT VII – GENOMICS & CANCER Big Campbell – Ch 17, 18, 20 Baby Campbell – Ch 10, 11, 12 Hillis – Chp 12, 13,

X. GENOMICS, cont Transposons o DNA sequences that move from one location in the genome to another o “Jumping Genes”

X. GENOMICS, cont Human Genome Applications  SNPs – single nucleotide polymorphisms o Single base-pair that shows variation in a significant % of population o SNPs that alter the fragment length following exposure to restriction enzymes called RFLPs (restriction fragment length polymorphisms) o Genetic markers

X. GENOMICS, cont Human Genome Applications, cont  STRs – short tandem repeats o Short segments of DNA that are highly repetitive, polymorphic o Repeat patterns are inherited o Useful for DNA fingerprinting

XI. BIOTECHNOLOGY Tools Restriction Enzymes o Used by bacteria to “chop up” viral DNA o Bacterial DNA protected by _________ o Very specific  Each enzyme recognizes a particular nucleotide sequence  Called a restriction sequence or restriction site  Palindromic  Cuts made at specific points  May create “ blunt en ds” or “ sticky ends ” o Used in gel electrophoresis o Also used to form recombinant DNA  Fragments may be pasted together with DNA ligase to form recombinant DNA

XI. BIOTECHNOLOGY, cont Tools, cont Polymerase Chain Reaction (PCR) o In vitro method of amplifying small amounts of DNA  DNA is heated to separate the double helix.  Mixture is allowed to cool, DNA primers attach to target  Heat-stable polymerase is used to extend the primers in the 5’–3’ direction.

XI. BIOTECHNOLOGY, cont Tools, cont Gel Electrophoresis o Separates DNA fragments based on size o Restriction fragment analysis  DNA treated with restriction enzymes  Resulting fragments migrate based on size  Produce a pattern characteristic of original DNA and restriction enzyme used

XI. BIOTECHNOLOGY, cont Tools, cont Southern Blotting  Designed by Dr. Southern  Detects particular DNA sequences Northern Blotting  Detects particular mRNA sequences Western Blotting  Used to detect proteins

XI. BIOTECHNOLOGY, cont Tools, cont cDNA - complementary DNA o Procedure for “cloning DNA” that uses mRNA, reverse transcriptase o

XI. BIOTECHNOLOGY, cont Recombinant DNA  DNA containing nucleotides from other sources  Process utilizes restriction enzymes that make jagged cuts in DNA; creates sticky ends  When DNA from different sources treated with same restriction enzyme, sticky ends “mix & match”  Often use reporter genes to determine success; for example, ampicillin resistance

XI. BIOTECHNOLOGY, cont DNA Microarray Assays o AKA DNA Chips o Test used to determine gene function, gene interactions o May be used to determine agressiveness of cancers, method of treatment, etc

XI. BIOTECHNOLOGY, cont Gene Cloning  Typically plasmid vector  Plasmid isolated from bacterial cell  Foreign DNA inserted into plasmid  Plasmid returned to bacterial cell; described as recombinant bacterium  Foreign gene is cloned as bacteria reproduce  Common bacterium used for plants is Agrobacterium tumefactiens

XI. BIOTECHNOLOGY, cont A CLOSER LOOK AT GENE CLONING

XI. BIOTECHNOLOGY, cont Therapeutic Cloning  ES  iPS

XI. BIOTECHNOLOGY, cont Reproductive Cloning  Nuclear Transplantation  Process of using unfertilized egg cell & replacing nucleus with DNA  In 1997, scientists were able to produce first reproductive clone, “Dolly”, by culturing somatic cells in a nutrient-poor medium to de-differentiate them and force them back to totipotency.  Reproductive cloning in animals has enjoyed limited success.

XI. BIOTECHNOLOGY, cont Gene Silencing o Knockout Genes  Use of genetic recombination to create an inactive, “knocked out” gene  Mutated allele introduced into embryonic stem cells  Forms chimeras  Often used in mice to study gene expression

XI. BIOTECHNOLOGY, cont Transgenic Organisms

XI. BIOTECHNOLOGY, cont o RNAi  Based on principal of microRNA  Exogenous small- interfering RNA (siRNA) synthesized taken up by cells or gains entrance via a viral vector  Complementary to mRNA target  Translation is blocked  Has been used to block production of growth factors in certain cancers

X. A CLOSER LOOK AT CANCER In early 1900s, scientists realized there are viruses that can cause cancer, including Human Papilloma virus, Epstein-Barr virus, and HTLV. Research led to discovery of cancer-causing genes called oncogenes We now know there are two important categories of genes in which mutations may lead to cancer o Oncogenes/Proto-oncogenes - arises from a genetic change that leads to an increase in either the amount of the proto-oncogene’s protein product or in the activity of each protein molecule. o Tumor Suppressor Genes - these genes normally produce products that inhibit cell division to prevent uncontrolled growth.

X. A CLOSER LOOK AT CANCER, cont Oncogenes  Amplification – Increases number of copies of proto-oncogene; will increase protein production  Point mutation in the promoter for an proto-oncogene, or in the gene itself  Movement of DNA - May change the rate at which gene at which gene is transcribed, therefore, translated  Translocation  Transposons  “Jumping Genes”  Genes that are moved due to folding of DNA, cut (or copy) & paste mechanism

X. A CLOSER LOOK AT CANCER, cont

Tumor-Suppressor Genes o Encode for proteins that inhibit cell division therefore any mutation that inhibits activity of tumor-suppressor gene may lead to abnormal cell growth and formation of tumors. o Act by producing proteins that repair damaged DNA, control density- dependent inhibition & anchorage dependence, or act as CDKs o Gene that is most often defective in human cancers codes for transcription factor known as p53  Known as the “guardian angel of the genome”  Serves as the master brake on the cell cycle when DNA damage has occurred

X. A CLOSER LOOK AT CANCER, cont Tumor Suppressor Genes, p53 cont.  When stimulated by DNA damage, p53 activates several genes with multiple effects  Genes activated to halt cell cycle  DNA repair genes turned on  If DNA damage cannot be repaired, “suicide genes” are activated; results in apoptosis

X. A CLOSER LOOK AT CANCER, cont

Tumor-Suppressor Genes, cont o BRCA 1, BRCA 2 genes o BRCA 1 Women who inherit one mutant allele have ~ 60% chance of having breast cancer by 50 Individuals with two normal alleles have ~ 2% chance

X. A CLOSER LOOK AT CANCER, cont