Term
| describe the numbers of the human genome sequence |
|
Definition
| 3 billion base pairs, 20000-25000 genes due to alternative splicing |
|
|
Term
| currently, what is the human genome sequence used for |
|
Definition
|
|
Term
| what are the main achievements currently in bio technology |
|
Definition
| restriction endonucleases, DNA sequence, cloning DNA, creation of synthetic probes, PCR |
|
|
Term
| what is another name for a restriction enzyme |
|
Definition
|
|
Term
| what does a restriction enzyme do |
|
Definition
| cleave specific DNA sequences |
|
|
Term
|
Definition
| a 4-8 base pair sequence that reads the same 5'-3' on both strands and is cleaved by an endonuclease |
|
|
Term
| what is the result after a restriction enzyme does his job |
|
Definition
|
|
Term
|
Definition
| overlaping sequence made by a restriction enzyme (zipper like) |
|
|
Term
|
Definition
| direct cut of a DNA sequence by a restriction enzyme (not zipper like) |
|
|
Term
| after a restriction enzyme cleaves, how is it possible that the area can be ligased if it was ruiened |
|
Definition
| 3' OH and 5' phosphate are attached after clevage |
|
|
Term
| what is a restriction site |
|
Definition
| the sequence a restriction enzyme is cleaved, the palendrome |
|
|
Term
| describe the relationship between the recognition sequence length and the frequency of DNA cuts |
|
Definition
| the shorted the restriction site the more frequent it will be |
|
|
Term
|
Definition
| a compliation of cleaved restriction sites that have been ligated |
|
|
Term
| what type of restriction site cleavage is easier to work with |
|
Definition
|
|
Term
| describe the basic concept of DNA cloning |
|
Definition
| a restriction sequence is inserted into a cloning vector in host cells, DNA is cloned by the cell and amplified making recombinent DNA |
|
|
Term
|
Definition
| a DNA molecile that accepts foriegn DNA fragments |
|
|
Term
|
Definition
|
|
Term
| what are the requirements of a vector for it to work with DNA cloning |
|
Definition
| autonomous replicationin the cell (so it needs an replication origin sequence), at least one restriction site, at least 1 gene for selection |
|
|
Term
| describe what a selection gene is and why it is needed |
|
Definition
| it ia gene within a vector that codes for some sort of selection, like an antibiotic resistance, because not all cells will have a vector with the restriction sequence in it and you need to weed out the ones that dont |
|
|
Term
| what are the common host cells for vectors |
|
Definition
| bacteria, yeast artificial chromosomes, retroviruses, yeast, phages |
|
|
Term
|
Definition
| a virus that infects bacteria |
|
|
Term
| what is a mammalian virus |
|
Definition
|
|
Term
| what are the types of DNA libraries |
|
Definition
|
|
Term
| what is the process of creating a genomic DNA library |
|
Definition
| take DNA from an organism, chop it up with restriction enzymes, ligate to a vector, let the host make many copies of each gene piece |
|
|
Term
| what does a genomic DNA library contain in the end |
|
Definition
| all sequences in the genome: introns, exons, promoters, etc |
|
|
Term
| what is the basic definition of a cDNA library |
|
Definition
| DNA compliment of mRNA that gives a snap shot of what was going on in that cell at that time |
|
|
Term
| describe the process of making a cDNA library |
|
Definition
| get a particular mRNA sequence from a cell, use reverse transcriptase to get a single DNA strand, use DNA polymerase to make it a double strand, put DNA into a vector and allow it to replicate |
|
|
Term
| in the end what does a cDNA library contain |
|
Definition
| no promoters, no introns, only mRNA |
|
|
Term
| how can a cDNA library be used after it has been made |
|
Definition
| put DNA clones into an expression vector to make mRNA then make protein |
|
|
Term
| describe the process of DNA sequencing |
|
Definition
| divide ssDNA, dNTPs, primers, and polymerases into 4 tubes into 4 tubes, add a specific dideoxyribonucleotide to each tube, synthesis proceedes until a dNTP is added in each strand, gel elecrtophoresis divides by length of products |
|
|
Term
| what do you need to do DNA sequencing |
|
Definition
| ssDNA, dNTPs, primer, polymerase |
|
|
Term
| what does DNA sequencing accomplish |
|
Definition
| determines the exact sequence of cloned DNA |
|
|
Term
|
Definition
| to identify DNA fragments |
|
|
Term
|
Definition
| ssDNA labeled (radioactivly usually) that can be hybrixised to ssDNA that is complimentary |
|
|
Term
|
Definition
| when target DNA is made single stranded by a method like heat or chemicals |
|
|
Term
| if probes use ssDNA how do they not reanneal |
|
Definition
| nitrocellulose membrane solid supports |
|
|
Term
| what happens when a nitrocellulose membrane is exposed to a probe |
|
Definition
| if complimentary, probe will bind and can be identified by autotraiography |
|
|
Term
| how long is a small probes |
|
Definition
|
|
Term
| how are small probes made |
|
Definition
| chemically synthesized oligonucleotides the same way synthetic primers are made |
|
|
Term
| what is the purpose of small probes |
|
Definition
| very specific, can identify a single base pair mutation |
|
|
Term
| how are large probes made |
|
Definition
| reverse transcription, PCR, etc. |
|
|
Term
| what is the function of large probes |
|
Definition
| can identify similar genes in different organisms or the same gene in different indiviguals that may not be exactly the same sequence |
|
|
Term
| what does southern blotting analize |
|
Definition
|
|
Term
| describe the process of southern blotting |
|
Definition
| isolate DNA, chop it with restriction enzymes, gel electrophotesis, denature DNA, blott it to immobilize it on the membrane, probe the blot |
|
|
Term
| what does southern blotting focus on |
|
Definition
|
|
Term
| what does northern blotting target |
|
Definition
|
|
Term
| describe the requirements for northern blotting |
|
Definition
| do not need to make a single stranded, probe must be complimentary to the mRNA |
|
|
Term
| what does northern blotting specifically detect |
|
Definition
|
|
Term
|
Definition
| tissue or cell specific studies, measure gene expression |
|
|
Term
| what does a western blot target |
|
Definition
|
|
Term
| what is the probe in a western blot |
|
Definition
| antibody specific to the protein of interest attached to an enzyme |
|
|
Term
| what is the function of a western blot |
|
Definition
| quantative, tells how much protein you have |
|
|
Term
| what is a restriction fragment length polymorphism (RFLP) |
|
Definition
| genetic differences due to polymorphisms in one of the 99.9% noncding regions that are inheriently not harmful or do not containa phenotype |
|
|
Term
| what are the requirements to be considered a RFLP |
|
Definition
| create or deletes a restriction site, has more or less of a type of repeated sequence |
|
|
Term
| what are the causes of RFLP |
|
Definition
| single nucleotide polymorphisms, disease causing mutation, harmless changes, tandem repeats |
|
|
Term
| what can single nucleotide polymorphisms (SNPs) cause |
|
Definition
| create of abolish a restriction site, 90% of the genetic variation in humans |
|
|
Term
| what does a variable number of tandem repeats (VNTR) refer to? |
|
Definition
| human genome contains many regions where a sequence is repeated intandem many times that varies greatly from person to person that are not related and some between people related |
|
|
Term
| physycally, how is a RFLP produced |
|
Definition
| DNA is cleaved on either side of a VNTR |
|
|
Term
|
Definition
| single nucleotide change that makes or abolishes a restriction site messing p the action of the restriction enzyme giving different sized fragments when run on a gel |
|
|
Term
| compared to VNTR describe the prevlience of SNP in the genome |
|
Definition
| SNPs are distributed through out |
|
|
Term
|
Definition
| to locate a diseased gene (not that it is the disease causing mutation but they tend to be near them), to mark allales (disease markers) |
|
|
Term
| what does it mean that a tandem repeat is hypervariable |
|
Definition
| different in all people especially in those not related |
|
|
Term
|
Definition
| not associated with disease, for paternity testing, forensics, molecular finger print |
|
|
Term
| what do you need to know to do a PCR |
|
Definition
| the flanking sequence around the sequence you want to amplify |
|
|
Term
| what are the advantages of PCR |
|
Definition
| amplify small amounts of DNA many times in a few hours, all in one test tube, DNA can be used for many reasons, amplify mutations to learn sequence, detect latent viruses, forensics, safer amniocentesis |
|
|
Term
| describe the process of PCR |
|
Definition
1. design primer to find flanking sequences,
2. denature DNA to make ssDNA using heat close to water boiling point
3. add primer to get DNA polymerase started, cool a bit so primer can anneal
4. chain extension using dNTPs and DNA polymerase
5. repeat steps 2-4 20-30 times |
|
|
Term
| what is a flanking sequence |
|
Definition
| approx 20 base pairs on each DNA strand before the DNA sequence you want to do PCR on so a primer can identify it |
|
|
Term
| in PCR, when you cool after denaturing so the primer can bind, does the DNA strand not just re-anneal |
|
Definition
| because we add lots of primer to make that unlikley and the cooling is very fast |
|
|
Term
| what happens if in PCR the middle part of the DNA strand (between the primers) reanneals during cooling |
|
Definition
| DNA polymerase will push it back open when it comes by |
|
|
Term
|
Definition
| part of its life it is hidden in the genome at low levels, can be found with PCR |
|
|
Term
| how does PCR help forensics |
|
Definition
| small sample size is ok now, just amplify |
|
|
Term
| how did PCR make prenatal genetic testing safer |
|
Definition
| during amniocentesis, smaller sample size is used because we can just amplify, less invasive |
|
|
Term
| how can we assess mRNA levels |
|
Definition
| northern blot, microarray |
|
|
Term
| how are the results of a northern blot interperted |
|
Definition
| band = yes the mRNA was expressed, band width tells quantity |
|
|
Term
| what does a microarray show |
|
Definition
| mRNA expression for 1000s of genes at a time |
|
|
Term
| how does a microarray work |
|
Definition
| glass slide with 1000s of divits, each divit has ssDNA with a compliment to gene with bases pointed outwards, isolate the mRNA sample and make cDNA copy, if compliment is on slide it will bind to it. always comparing 2 samples |
|
|
Term
| how do you read microarray results |
|
Definition
yellow: samples are equal in expression
black: only one sample had expression
red: one sample expressed more
green: the other sample expressed more |
|
|
Term
| what reads the results of a microarray |
|
Definition
| a machine that can analize the exact shades and determine conecntrations |
|
|
Term
| what does proteomics evaluate |
|
Definition
| proteins made in a cell, post translational modifications, turn over of proteins, tissue comparison, enzyeme modulations |
|
|
Term
| what does ELIZA stand for |
|
Definition
| enzyme linked ammunosorbent assay |
|
|
Term
|
Definition
| protein is linked to an enzume and put in a 96 microwell plate, antigen is bound to plate well, probe with antibody is linked to the enzyme, add colored substrate to see how much protein bound, bound protein = protein made in cell |
|
|
Term
| how do you read the results of a western blot |
|
Definition
| gives color reaction and exact band size |
|
|
Term
| what protein / DNA expression techniques use a gel |
|
Definition
| southern, northern, and western blot, proteomics, PCR (maybe) |
|
|
Term
| what protein / DNA expression techniques are quantative |
|
Definition
| norther and western blots, microarray, elisa, proteomics, PCR sometimes |
|
|
Term
| what protein is affected by a sickle cell anemia mutation and how |
|
Definition
| b-globin by eliminating a restriction site |
|
|
Term
| what type of mutation is involved in sickle cell anemia |
|
Definition
| a point mutation creating a RFLP |
|
|
Term
| why is sickle cell anemia a special type of RFLP |
|
Definition
| because it is one of the few times where the RFLP mutation is disease causing |
|
|
Term
| what are the ways you can test for sickle cell anemia |
|
Definition
| PCR, southern blot, allele specific olegonucleotide probes |
|
|
Term
| explain how to interpert the results of a southern blot for sickle cell anemia |
|
Definition
| sickle cell has one larger (higher on gel) band, a carrier has two bands, a normal allele will have one smaller band |
|
|
Term
| what type of inheritence does sickle cell have |
|
Definition
| recessive, a heretozygote will have no symptoms |
|
|
Term
| explain the process of doing a PCR to detect sickle cell anemia |
|
Definition
| design a primer to flank the B-globin gene, amplify the mutation region, digest the fragment with a restriction enzyme and run a gel |
|
|
Term
| where does the specificity come from when running a PCR to determine sickle cell anemia |
|
Definition
| designing a primer for the mutation region |
|
|
Term
| describe how to read the results for a PCR on sicle cell anemia |
|
Definition
| there will be one larger (higher on gel) band for sickle cell and two smaller bands for a normal patient |
|
|
Term
| describe the process of allele specific olegonucleotide probing |
|
Definition
| get samples from people, make two wells per person, probe one well with the normal gene and one with the mutated gene, add in the samples, see which well has the reaction |
|
|
Term
| what types of mutations can allele specific olegonucleotide probing find |
|
Definition
|
|
Term
| what gene is mutated in cystic fibrosis, what does this gene have a role in |
|
Definition
|
|
Term
| what is the most common lethal genetic mutation in caucasions |
|
Definition
|
|
Term
| what are some of the symptoms of cystic fibrosis |
|
Definition
| chloride in sweat, lack of chloride secretion in the lungs leading to infection and mucus build up, build up of mucus in the pancreas, death around age 30 |
|
|
Term
| what type of mutation affects most people with cystic fibrosis, what amino acid is missing due to it |
|
Definition
|
|
Term
| what test do we use to determine cystic fibrosis |
|
Definition
|
|
Term
| what type of inheritence is cystic fibrosis |
|
Definition
| recessive, need two mutant genes to get the symptoms |
|
|
Term
| describe how to do a PCR to test for cystic fibrosis |
|
Definition
| possible deletion area is flanked making different size products depending on if the deletion area is there or not |
|
|
Term
| describe how to interpert the results of a PCR for cystic fibrosis |
|
Definition
| the mutant will have one smaller band (it weights less because of the cut due to the mutation), the normal will have one larger band, a carrier will have both bands |
|
|
Term
| describe the process of allele specific olegonucleotide probing |
|
Definition
| get samples from people, make two wells per person, probe one well with the normal gene and one with the mutated gene, add in the samples, see which well has the reaction |
|
|
Term
| what types of mutations can allele specific olegonucleotide probing find |
|
Definition
|
|
Term
| what gene is mutated in cystic fibrosis, what does this gene have a role in |
|
Definition
|
|
Term
| what is the most common lethal genetic mutation in caucasions |
|
Definition
|
|
Term
| what are some of the symptoms of cystic fibrosis |
|
Definition
| chloride in sweat, lack of chloride secretion in the lungs leading to infection and mucus build up, build up of mucus in the pancreas, death around age 30 |
|
|
Term
| what type of mutation affects most people with cystic fibrosis, what amino acid is missing due to it |
|
Definition
|
|
Term
| what test do we use to determine cystic fibrosis |
|
Definition
|
|
Term
| what type of inheritence is cystic fibrosis |
|
Definition
| recessive, need two mutant genes to get the symptoms |
|
|
Term
| describe how to do a PCR to test for cystic fibrosis |
|
Definition
| possible deletion area is flanked making different size products depending on if the deletion area is there or not |
|
|
Term
| describe how to interpert the results of a PCR for cystic fibrosis |
|
Definition
| the mutant will have one smaller band (it weights less because of the cut due to the mutation), the normal will have one larger band, a carrier will have both bands |
|
|
Term
| what type of inheritence is PKU |
|
Definition
|
|
Term
| why are all newborns screened for PKU |
|
Definition
| because the symptoms can be avoided with a special diet |
|
|
Term
| what process is inhibited during PKU |
|
Definition
| phenylalanine turning into tyrosine |
|
|
Term
| why can we use ASO probing or PCR to find PKU |
|
Definition
| because there are over 400 mutation site possibilities and you would have to make over 400 primers with special flanking regions or have over 400 wells for ASO |
|
|
Term
| what are the symptoms of PKU |
|
Definition
|
|
Term
| how many exons could have a PKU causing mutation |
|
Definition
|
|
Term
| what types of mutations could cause PKU |
|
Definition
| mostly missense and some splice, nonsense, insertions, deletions |
|
|
Term
| describe how to do RFLP analysis to determine PKU |
|
Definition
| collect DNA from many family members including 1 person with the disease and the patient, find a RFLP marker that is near the disease site, do a southern blot on the RFLP marker and compare the patient with the normal, carriers, and affected family members to find the patient's result |
|
|
Term
| what kind of mutation cause myotonic dystrophy and of what gene |
|
Definition
| 3' non-coding trinucleotide repeat of a protein kinase gene |
|
|
Term
| what is the most common adult muscular dystrophy |
|
Definition
|
|
Term
| describe how to do RFLP comparison to determine muscular dystrophy |
|
Definition
| digest part of the RFLP and get an identifiable sequence, compare to family members who are affected or not, the mutant allele may be different in each person but you compare to the family members so you can see what normal looks like in that family |
|
|
Term
| what happens to trinucleotide repeats over time |
|
Definition
| they get bigger with each generation |
|
|
Term
| why can we do PCR on a trinucleotide repeat |
|
Definition
| PCR can flank the expansion region but when it gets too big it becomes difficult for PCR to amplify the region |
|
|
Term
| what techniques do you use to detect HIV |
|
Definition
| immunoassays: ELIZA and western blot |
|
|
Term
| why is it difficult to detect HIV early |
|
Definition
| because it takes years for the symptoms to develop because it takes a long time for antibodies to form |
|
|
Term
| at what point can you test for HIV |
|
Definition
| around 6 months after infection there should be enough antibodies, but you can do it earlier but retest after 6 months |
|
|
Term
| how to use ELIZA to test HIV |
|
Definition
| bind proteins to the wells and add the HIV antibody, add the sample to the wells, if there is an HIV antibody there will be a reaction causing color change |
|
|
Term
| why do we also do a western blot to test for HIV |
|
Definition
| ELIZA is super sensitive and could give a flase positive so you want to test the protein to make sure it is the right size and is HIV |
|
|
Term
| how do you do a western blot to test for HIV |
|
Definition
| do electrophorsis to seperate sample, probe for a protein reaction and verify the protein by size |
|
|
Term
| how has PCR revolutionized HIV testing |
|
Definition
| test can be done immediatly because you need less sample, you can PCR for the provisus to test time now, you can do reverse transcription PCR for HIV itself, you can monitor HIV over time (quantative) |
|
|
Term
| how is paternity testing done |
|
Definition
| design a primer to flank VnTR molecular fingerprint and amplify, stain for any DNA present (no probe) and compare to family |
|
|
Term
| what is the paternity index |
|
Definition
| because VNTRs are not perfect between family members different states require you to test a different amount of VNTRs before making a decision on paternity |
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