1. Ch 13: genetic engineering
1.1. DNA cloning
1.1.1. nucleic acid hybridization
1.1.1.1. the base paring of one strand of a nucleic acid to a complementary sequence on another strand
1.1.2. genetic engineering definition
1.1.2.1. the direct manipulation of genes for practical purposes
1.1.3. eukaryotic genomes: proportion of genes vs noncoding nucleotide sequences
1.1.3.1. small proportion of genes
1.1.4. plasmids
1.1.4.1. small, circular DNA molecules that replicate separately from the bacterial chromosome
1.1.5. DNA Cloning process
1.1.5.1. 1. obtain plasmid
1.1.5.2. 2. insert DNA
1.1.5.2.1. recombinant DNA
1.1.5.3. 3. plasmid returned to bacterial cell
1.1.5.3.1. produces recombinant bacterium
1.1.5.4. 4. bacterium reproduces
1.1.6. basic purposes
1.1.6.1. make many copies of particular gene (amplify)
1.1.6.2. produce a protein product
1.2. Restriction enzymes
1.2.1. def: enzymes that cut DNA molecules at a limited number of specific locations
1.2.2. restriction endonucleases
1.2.3. in bacteria
1.2.3.1. protect cell by cutting up foreign DNA
1.2.4. restriction site
1.2.4.1. particular short DNA sequence the restriction enzyme recognizes and cuts
1.2.4.2. most are symmetric
1.2.4.2.1. example: GAATTC paired to CTTAAG
1.2.5. restriction fragment
1.2.5.1. DNA molecule produced by a restriction enzyme
1.2.6. sticky end
1.2.6.1. restriction fragment end that is single-stranded
1.2.6.2. allows them to hybridize to sticky ends on other DNA molecules cut with the same enzyme
1.2.7. cloning vector
1.2.7.1. a DNA molecules that can carry a foreign DNA into a host cell and replicate there
1.3. PCR
1.3.1. polymerase chain reaction
1.3.2. process
1.3.2.1. 3 step cycle
1.3.2.1.1. 1. denaturation: separate DNA strands
1.3.2.1.2. 2. annealing
1.3.2.1.3. 3. extension
1.3.3. key in automation
1.3.3.1. discovery of unusually heat-stable DNA polymerase
1.3.3.1.1. Taq polymerase
1.3.4. key to specificity
1.3.4.1. primers hybridize only with complementary sequences at opposite ends of the target segment
1.3.5. cannot substitute for gene cloning in cell
1.3.5.1. occasional errors during PCR replication limit the number of good copies and the length of DNA fragments that can be copied
1.3.5.2. PCR used to provide DNA for cloning
1.4. DNA sequencing
1.4.1. determine gene's complete nucleotide sequence
1.4.2. sequencing by synthesis of the complementary strand one nucleotide at a time
1.5. Antibiotic resistance in plasmid
1.5.1. helps scientists distinguish between bacteria with and without the plasmid
1.6. recombinant DNA tech & Medical applications
1.7. antagonistic hormones
1.7.1. maintain homeostasis
1.7.2. example: insulin and glucagon
1.7.2.1. blood sugar level increases, insulin created to stimulate cells to absorb glucose, blood sugar level drops, glucagon stimulates liver to release glucose
2. Central Dogma and Genetic Medicine (tinyurl.com/apbcdagm)
2.1. Level of DNA
2.1.1. CRISPR-Cas9
2.1.1.1. 2 main uses
2.1.1.1.1. knock out a gene
2.1.1.1.2. edit a gene to correct a disease-causing mutation
2.1.1.2. components
2.1.1.2.1. nuclease (Cas9)
2.1.1.2.2. guide RNA
2.1.1.3. clustered regularly interspaced short palindromic repeats
2.1.2. Gene Therapy
2.1.2.1. Leber Congenital Amaurosis
2.1.2.1.1. Viral Vectors
2.1.2.1.2. causes extreme far-sightedness or blindness at birth
2.1.2.1.3. mutations disrupt processes by which cells in retina convert light into nerve signals to the brain
2.1.2.1.4. gene therapy
2.2. Level of DNA Transcription
2.2.1. Gene Switches
2.2.1.1. regulatory DNA sequences in genome
2.2.1.2. switch sequences are noncoding and regulatory proteins can bind to them, turning on or off transcription
2.2.1.3. Sickle cell disease
2.2.1.3.1. caused by mutations in gene for beta-globin
2.2.1.3.2. fetal hemoglobin
2.3. Level of RNA Splicing
2.3.1. exon skipping
2.3.1.1. changes how the primary RNA transcript is spliced, removing mutation
2.3.1.2. short segment of single-stranded RNA (antisense RNA)
2.3.1.3. antisense RNA causes splicing machinery to skip over a segment of the transcript
2.3.1.4. one or more exons are spliced out
2.3.1.5. the mRNA produces a shortened, partially functional protein instead of a severely truncated or absent protein
2.3.2. Duchenne muscluar dystrophy
2.3.2.1. mutation in gene that codes for protein dystrophin
2.3.2.1.1. gene that codes for dystrophin is one of the largest human genes
2.3.2.1.2. mutations can also cause becker MD
2.3.2.2. exon-skipping drug eteplirsen can cause mutatino to be spliced out of dystrophin mRNA
2.4. Level of mRNA Transport
2.4.1. RNA interference
2.4.1.1. involve small RNA segments that target mRNAs for destruction (reduces expression of genes)
2.4.1.2. RNAi method
2.4.1.2.1. uses small interfering RNA (siRNA)
2.4.1.2.2. double-stranded siRNA taken up by cells and cleaved into smaller pieces (bc seen as foreign)
2.4.1.2.3. single RNA strands from siRNA pieces are then incorporated into cellular protein complex (RNA-induced silencing complex (RISC))
2.4.1.2.4. siRNA guides RISC to mRNA with complementary sequence and cleaves it
2.4.1.3. siRNA and microRNA (miRNA) can be used
2.4.1.3.1. siRNA
2.4.1.3.2. miRNA
2.4.1.4. cells produce miRNAs for regulating gene expression
2.4.1.5. cells produce siRNAs as a defense mechanism against double-stranded RNA genomes of some viruses
2.4.2. Huntington's disease
2.4.2.1. caused by mutations in gene called hintintin (HTT), which is required for normal nerve cell function
2.4.2.1.1. mutations--> abnormal protein--> brain cells die over time
2.4.2.2. symptoms start in adulthood and worsen over time
2.4.2.2.1. dementia, gradually deteriorating motor function, death
2.4.2.3. involves mutation with CAG repeats
2.4.2.3.1. usually 10-35, but with HD, there are more and proteins have abnormally long stretches of glutamines that cause proteins to stick together and accumulate in nerve cells
2.4.2.3.2. 36-39 repeats may or may not develop symptoms
2.4.2.3.3. 40+ repeats almost always develop symptoms
2.5. Level of protein processing
2.5.1. small molecule drugs
2.5.1.1. bc small size, easily taken up by cells and administered to patients
2.5.1.2. some directly interact with disease-causing proteins, others through other molecule, some block negative effects of disease-causing proteins, some restore proper function
2.5.2. cystic fibrosis
2.5.2.1. caused by mutations in the CFTR gene that codes for cystic fibrosis transmembrane conductance regulator (CFTR)
2.5.2.1.1. channel for Cl- ions
2.5.2.1.2. lead to cells producing thick, sticky mucus
2.5.2.1.3. most common mutation: F508del
2.5.2.2. small molecule lumacaftor improves processing of mutant CFTR protein, increasing amount of protein in cell membrane
2.5.2.3. small molecule ivacaftor increases time CFTR channels remain open
2.5.2.4. makes person susceptible to lung infections
2.5.2.5. includes info from: tinyurl.com/apbcysfib
3. Cancer and Cell cycle and tumor growth (tinyurl.com/apbcacc)
3.1. review cell cycle phases from last semester
3.1.1. AP Bio Semester 1
3.1.2. under "Mitosis and Meiosis"
3.2. cell cycle regulators and cancer
3.2.1. genes that encode for cell cycle regulators (stimulate or inhibit cell cycle progression)
3.2.1.1. proto-oncogenes
3.2.1.1.1. encode for stimulating proteins
3.2.1.2. tumor suppressor genes
3.2.1.2.1. encode for inhibitory protens
3.2.2. most important: cyclin-dependent kinases
3.2.2.1. kinases add phosphates to other proteins to activate/inhibit
3.2.2.2. always present in cell but becomes active when bound to cyclins
3.2.3. G1 checkpoint
3.2.3.1. inhibiting proteins
3.2.3.1.1. tumor suppressor genes
3.2.3.2. stimulating proteins
3.2.3.2.1. G1 CDK-cyclins
3.2.4. S checkpoint
3.2.4.1. inhibiting proteins
3.2.4.1.1. ataxia telangiectasia mutated protein
3.2.4.1.2. breast cancer 1 (BRCA1)
3.2.4.2. stimulating
3.2.4.2.1. CDK-cyclin complexes
3.2.5. G2 checkpoint
3.2.5.1. p53
3.2.6. M checkpoint
3.2.6.1. mitotic arrest deficient proteins
3.2.6.2. inhibits anaphase-promoting complex/cyclosome, preventing entry into anaphase
3.3. VEGF (tinyurl.com/vegfapb)
3.3.1. vascular endothelial growth factor
3.3.2. growth factor for blood vessel cells
3.3.3. blood vessels are stimulated to grow
3.3.4. for example, cancer cells may secrete this growth factor to cause blood vessels to grow toward the cancerous cells
3.4. tumor growth (tinyurl.com/tgapb)
3.4.1. cancerous cells keep dividing at the expense of normal cells sround them
3.4.2. once large enough to see with the naked eye, they recruit blood vessels (angiogenesis)
3.4.3. metastasis:
3.4.3.1. cells go in blood vessel to another part of the body, and form new cells there
4. CRISPR more indepth (tinyurl.com/crhiwapb)
4.1. targeting
4.1.1. nuclease - Cas9
4.1.1.1. recognizes and binds to PAM
4.1.1.1.1. 3-nucleotide motif
4.1.1.1.2. protospacer adjacent motif
4.1.1.1.3. occur usually every 50ish bases in humans
4.1.2. guide RNA
4.1.2.1. matches particular sequence of DNA
4.1.2.2. must be a sequence near a PAM motif
4.2. binding
4.2.1. after binding to motif, Cas9 unwinds and pulls apart DNA double helix upstream of PAM
4.2.2. if sequence is not exact match to guide RNA (usually 20 nucleotides) Cas9 disengages and DNA zips up
4.2.3. if sequence is perfect match, guide RNA base pairs with complementary DNA sequence forming DNA-RNA helix
4.3. cleaving
4.3.1. creation of DNA-RNA helix activates Cas9 nuclease, which cuts DNA 3 nucleotides upstream from PAM
4.3.1.1. both strands are cleaved: double stranded DNA break
4.4. DNA repair
4.4.1. NHEJ
4.4.1.1. nonhomologous end joining
4.4.1.1.1. more frequently used
4.4.1.1.2. error prone
4.4.1.1.3. Cas9 will cleave again if error repaired correctly
4.4.1.1.4. random mutation within desired target sequence
4.4.2. HDR
4.4.2.1. homology-directed repair
4.4.2.1.1. less error-prone
4.4.2.1.2. uses homologous DNA template to accurately repair break
4.4.2.1.3. scientists introduce excess of DNA repair template along with Cas9-guide RNA complex
4.4.2.1.4. repair template - change target DNA sequence or correct existing mutation by replacing it with a nonmutated sequence of DNA
4.5. how it's used
4.5.1. see website...
5. Allele Frequency
5.1. p^2 + 2pq + q^2 = 1
5.2. finding probability of allele match
5.2.1. homozygous: square the frequency
5.2.2. heterozygous: multiply the frequencies together and by 2
5.3. finding frequency of allele match
5.3.1. reciprocal of probability
6. Lactose Persistence/tolerance (tinyurl.com/ltapapb)
6.1. lactase digests lactose
6.1.1. active in infants
6.1.2. after weaning, humans stop producing lactase
6.2. mutation keeps lactase gene switched on past infancy
6.3. regulation of lactase gene
6.3.1. transcriptional regulation
6.3.1.1. not transcribing in the first place
6.3.1.2. transcription factors
6.3.1.2.1. general transcription factors
6.3.1.3. activators and repressors
6.3.1.3.1. activators
6.3.1.3.2. repressors
6.3.2. RNA Processing
6.3.2.1. alternative splicing:
6.3.2.1.1. different exons are spliced together
6.3.2.2. "inhibiting RNA processing is probably not a major way to regulate gene expression"
6.3.3. translational regulation
6.3.3.1. RNA interference
6.3.3.1.1. small pieces of RNA bind to mRNA to trigger degradation or block translation
6.3.4. protein processing and degradation
6.3.4.1. active proteins no longer needed/are damaged are marked for destruction
6.3.4.2. proteasomes
6.3.4.2.1. large protein complexes
6.3.4.2.2. recognize and degrade proteins that have been tagged with ubiquitin
6.4. populations from northern europe and regions in africa have mutations near LCT gene that causes lactase production to continue into adulthood
6.4.1. appeared at the same time as the domestication of cattle
6.4.2. mutations occur in the regulatory region
6.4.3. europe
6.4.3.1. mutation strengthens binding of transcription factor Oct-1 to enhancer region
7. DNA Sequence Assembly (tinyurl.com/dsaapb)
7.1. Sanger method (70s)
7.1.1. include a dideoxy terminator
7.1.1.1. do not include 3' hydroxyl so polymerase cannot extend beyond that point
7.1.1.2. fluorescently label them
7.1.2. you end up with molecules that are terminated at every base in the sequence (usually between 100-500 bases)
7.1.2.1. denature templates and separate the by size
7.1.2.2. upper limit is ~1000 nucleotides
7.1.3. as sequence chain lenthens, nucleotides differing by one nucleotide become harder to separate by size
7.2. reading a long dna sequence
7.2.1. sequence from partial 'read" used to prime next section of sequence
7.3. shotgun sequencing
7.3.1. break up long dna into smaller segments
7.3.2. each segment is sequenced
7.4. deep sequencing
7.4.1. hundreds of millions of short reads are generated simultaneously
7.4.2. chop DNA into small pieces
7.4.3. add primers
7.4.4. use a sequencing primer and extend with polymerase
7.4.5. use only terminators
7.4.6. reverse terminator (make it so it can be extended)
7.4.7. extend with another terminator
7.4.8. take image after every extention
7.4.9. extension, image, cleave, extension, image, cleave...
7.5. sequence assembly
7.5.1. for shotgun and deep sequencing, sequences must be put together to get the genome again.... like shredding multiple copies of a book and reassembling the original text by finding overlapping fragments
7.5.2. takes longer to assemble finished sequence than generate reads
7.5.3. some sequences may not be covered at all
7.5.3.1. use regerence genome to see where there are holes
7.5.3.2. finding gaps on sequence reads
7.5.3.2.1. sequences adjacent to the gaps are used to design primers to run PCR rxn to amplify viral DNA spanning gap
7.6. consensus sequence
7.6.1. is a way of showing the most common nucleotide at each position from multiple sequences that are similar enough to be aligned and compared
8. RNA Interference (tinyurl.com/RNAiapb)
8.1. experiment found that injecting double-stranded RNA turned down expression of genes with matching nucleotide sequence
8.1.1. experiment: scientists discovered that plants have RNA-dependent RNA polymerase (RDRP) that produces double-stranded RNA when triggered by very high levels of mRNA
8.1.1.1. RDRP created double-stranded RNA molecules that shut down gene expression and reduced pigment
8.2. RNA interference (RNAi)
8.2.1. the silencing of gene expression triggered by the presence of double-stranded RNA homologous to portions of the gene
8.2.1.1. can result from cleavage and degradation of target gene's mRNA
8.2.1.2. can result from blocking translation of intact mRNA
8.2.2. occurs in many animals
8.2.3. can protect the genome from some viruses and mobile genes
8.2.4. ex: guides embryo development by turning down specific genes at critical times
8.2.5. how it works
8.2.5.1. Dicer enzyme recognizes and cuts long double-stranded RNA
8.2.5.2. Dicer cleaves into siRNAs (small interfering RNAs)
8.2.5.3. siRNAs bind to proteins to form an assembly called the RNA-induced silencing complex (RISC)
8.2.5.4. RISC becomes activated when double-stranded siRNA it contains unzips (requires ATP)
8.2.5.5. once activated, RISC recognizes and binds to target mRNA
8.2.5.6. subunits of RISC cleave mRNA
8.3. why turn down genes
8.3.1. find function of gene
9. Genes as Medicine (Molly) (tinyurl.com/gamapb)
9.1. molly inherited a mutation ...
9.1.1. in the same gene from each of her parents
9.2. why are eyes good targets for clinical trials
9.2.1. easy to access
9.2.2. one can be treated and the other can be the control
10. Genetic Mutations and Disease (tinyurl.com/gmdapb)
10.1. germline
10.1.1. lineage of cells that gives rise to an individual's gametes
10.2. somatic cells
10.2.1. cells in the body that are not part of the germline
10.2.2. mutations in these will not be inherited by offspring
10.3. inherited mutations
10.3.1. all offspring's cells carry the mutation
10.3.2. example: cystic fibrosis and huntington disease
10.4. new mutations in germline
10.4.1. if a mutation occurs during gamete production, offspring may inherit mutation from unaffected parent (de novo mutation)
10.5. somatic cell mutations in development
10.5.1. occur relatively early in individual's life and can affect development of tissues and organs
10.6. somatic cell mutations later in life (cancer)
10.6.1. dont typically affect individual but can lead to cancer
10.6.2. cancer is not inherited
10.6.2.1. but individuals can inherit mutations that increase their risk of developing cancer
10.6.2.1.1. example: inherit mutations in BRCA1 gene are more likely to develop breast and ovarian cancer