Term
| What five components does DNA sequencing require? |
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Definition
1. DNA template strand
2. dNTPs
3. primer to initiate
4. DNA polymerase
(above same as DNA replication)
5. ddNTPs that terminate |
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Term
| How does a ddNTP cause termination of DNA synthesis? |
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Definition
| There is no free 3' OH to attack |
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Term
| Describe automated DNA sequencing |
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Definition
| The ddNTPs are flourescently colored so a computer can read the results |
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Term
| What sort of polymerase is used for PCR and why? |
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Definition
| A thermally stable one such as Taq polymerase (from thermophilic bacteria) that will function at the temperatures which cause DNA denaturation |
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Term
| What are the repeated steps of PCR? |
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Definition
| 1. DNA denaturation at high temperatures
2. Lowering temperature so two primers can anneal to the Tm on each template strand
3. Synthesis from the primers using Taq DNA polymerase |
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Term
| How can a sequence be added via PCR for cloning? |
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Definition
| A tail is added to a 5' end of a primer and it becomes incorporated when the primer adds the new restriction site |
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Term
| What are the two types of cleaved ends a recognition sequence can produce in cloning? Which are more effective? |
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Definition
Sticky ends and blunt ends
Ligands can still bind to blunt ends, but sticky ends are more effective |
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Term
| Describe the process of viral plasmid DNA cloning |
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Definition
1. Restriction enzymes cleave recognition sequences
2. New DNA fragments are ligated to the prepared cloning vector
3. This recombinant vector DNA is introduced into the host cell
4. The host cell propagates, producing many copies |
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Term
| What can cause errors in nucleic acids? |
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Definition
| Replication mistakes, spontaneous sequence changes (rare), and actions of mutagens |
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Term
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Definition
| Heritable changes in the DNA sequence |
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Term
| What two ways can a mutation arise in a single cell? |
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Definition
1. Cell division (somatic mutations)
2. Reproduction (germline mutations) |
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Term
| What are the three types of mutations? |
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Definition
1. Silent (no AA change)
2. Nonsense (AA changes to STOP codon)
3. Missense (AA changes to different AA) |
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Term
| Give an example of a genetic disease caused by a single missense mutation |
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Definition
Sickle-cell anemia
GAG (glutamate) changed to GTG (valine) in Beta-globin protein in red blood cell |
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Term
| What are the two types of spontaneous base changes? |
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Definition
1. Spontaneous loss of amino groups
2. Spontaneous depurination |
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Term
| What are the two types of spontaneous base changes? |
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Definition
1. Spontaneous loss of amino groups
2. Spontaneous depurination |
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Term
| What can cause spontaneous loss of exocyclic amino groups in bases? |
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Definition
C changes to U fairly frequently
Nitrates and nitrates can cause deaminations |
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Term
| What are the four examples of spontaneous loss of exocyclic amino groups? |
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Definition
1. Cytosine → Uracil
2. 5-Methylcytosine → Thymine
3. Adenine → hypoxanthine
4. Guanine → Xanthine |
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Term
| Describe spontaneous depurination |
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Definition
Occurs in 1/10,000 purines per cell per day
More frequent in DNA than RNA
Basically, a purine leaves the base leaving an apurinic reisude |
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Term
| What kind of base damage can UV light cause |
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Definition
Pyrimidine dimers that lead to problems in DNA replication
Ex. Covalently bonded cyclobutane thymidine dimer or a 6-4 diagonal photoproduct |
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Term
| What are some alkylating agents that can modify DNA? |
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Definition
S-Adenosylmethionine, Dimetyhlnitrosamine, Dimethylsulfate, Nitrogen mustard
(interesting trivia- these were all used as early chemotherapy chemicals) |
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Term
| How does Dimethylsufate (DMSO) affect DNA? |
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Definition
| It methylates guanine to O6-Methylguanine, which will bind to thymine instead of cytosine |
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Term
| Where are frameshifts most common? |
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Definition
| In sequences of repeating bases |
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Term
| What can frameshifts cause? |
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Definition
| Mistranslation, premature termination |
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Term
| Define deletion mutations |
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Definition
| Loss of DNA sequences (can be as much as thousands of bps) |
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Term
| Define insertion mutations |
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Definition
| Acquisition of a block of new or duplicated sequence |
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Term
| Define inversion mutations |
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Definition
| Reversal of sequence orientation |
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Term
| Define translocation mutations |
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Definition
| Change in location of a sequence to somewhere else on the same or different chromosome |
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Term
| Describe the Ames test for mutagens |
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Definition
1. The tested chemical is mixed (in increasing doses) with liver extract onto petri dishes
2. Histidine-requiring bacteria is spread on the histidine-free dishes
3. The bacteria that survive mutated to be able to synthesize histidine, if the chemical encouraged this it is mutagenic |
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Term
| Define Mismatch repair (MMR) |
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Definition
The major post-replication repair system
Phylogenetically conserved
Detects "bulges" where bases mispaired |
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Term
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Definition
| Most common post-replication repair, phylogenetically conserved, detects bulges where bases are mispaired |
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Term
| How does MMR recognize where it needs to work in gram negative bacteria? |
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Definition
MMR uses DNA methylation to discriminate between the methylated (old) template strand and the new strand
Methylation after replication is to distinguish it from invading bacterial DNA |
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Term
| What enzyme methylates gram (-) bacteria DNA after replication? |
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Definition
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Term
| How does MMR work in E. coli? |
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Definition
1. MutL and MutS proteins bind to the "bulge"
2. This complex binds to MutH
3. MutH binds to the closest methyl group, forming a large puckered region which is cleaved |
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Term
| What enzymes cleave out the large puckered area in MMR? |
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Definition
DNA exonuclease VII (5'→3')
or DNA exonuclease I (3'→5')
depending on where the methyl the MutH bound to is |
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Term
| What enzyme fills in the cleaved out area in MMR? |
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Definition
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Term
| In Gram + bacteria, how is the newly created DNA strand modified for mistakes? |
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Definition
| Exonuclease Exol modifies the polymerase III clamp (PCNA) |
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Term
| Describe the mechanism of Base Excision repair (BER) |
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Definition
| 1. A glycosylase enzyme cuts the base-sugar bond to remove a damaged or inappropriate base
2. AP endonuclease cleaves inside the strand
3. DNA polymerase I (5'→3') puts in the right base and ligase seals |
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Term
| Describe nucleotide excision repair (NER) |
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Definition
The major repair system in mammals for UV damage
Fixes a "bulky" area of DNA damage |
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Term
| Describe the mechanism for Nucleotide Excision Repair |
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Definition
1. Recognition of "bulky" DNA damage
2. Endonucleases cleave on either side of damage (around 30 bases)
3. DNA polymerase I fills in the gap
4. Ligase seals |
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Term
| How is NER different between mammals and bacteria? |
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Definition
| The only difference I can see is that the exinuclease cleaves out a larger area in mammals than in bacteria |
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Term
| Between MMR, BER, and NER which causes the largest excision and which causes the smallest? |
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Definition
| MMR is largest excised area, then NER, last BER only causes 1 base excised |
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Term
| Define xeroderma pigmentosum |
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Definition
Disease with defective nucleotide excision repair
Causes extreme sun sensitivity (since NER is the major repair system for UV damage) |
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Term
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Definition
| DNA photolyase fixes pyrimidine dimers by breaking the covalent bonds using light energy |
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Term
| How can O6-Methylguanine (caused by DMSO) be repaired? |
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Definition
| O6-Methylguanine metyltransferase removes the methyl group to a protein
This protein doesn't regenerate, which shows how important it is that the cell is willing to produce it just for this reaction |
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Term
| How can alkylated adenine or cytosine be repaired? |
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Definition
| AlkB enzyme (with Fe group) attaches an -OH to the unwanted methyl, then formaldehyde removes the H2C=O |
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Term
| Describe Recombinational (daughter-strand gap) repair |
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Definition
Generally accurate, recombination fixes a daughter-strand gap
Damage "tolerance" rather than repair
A good way to cope with a non-coding lesion |
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Term
| Give an example of a disease that may be caused by a defect in Recombinational (daughter-strand gap) repair |
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Definition
Human breast cancer
Susceptibility genes BRCA1 and 2 may be involved in repair |
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Term
| Define genetic recombination |
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Definition
| Rearrangement of genetic information within and among DNA molecules |
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Term
| What are the three types of genetic recombination? |
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Definition
1. Homologus recombination
2. Site-specific recombination
3. DNA transposition |
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Term
| Describe homologus recombination |
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Definition
Recombination between two identical or nearly identical DNA sequences
For both repair and for diversity in meiosis (crossing over) |
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Term
| Describe the mechanism for homologus recombination |
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Definition
| 1. A double strand break is converted to a gap by exonuclease, producing 3' single-strand extensions
2. An exposed 3' end pairs with its complement in the intact homolog, displacing the original
3. The invading 3' strand is extended by polymerase and branch migration producing crossovers called Holliday intermediates
4. Further DNA replication replaces missing DNA
5. Nucleases cleave Holliday intermediates, resulting in two diversified product sets |
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Term
|
Definition
The protein that helps initiate homologus recombination
Recognizes Chi sites and acts as an exonuclease to degrade |
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Term
| What does each section of RecBCD do? |
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Definition
RecB is a 3'→5' exonuclease
RecC recognizes Chi sites
RecD is a 5'→3' exonuclease |
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Term
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Definition
The protein that assembles and dissembles filaments in homologus recombination
ATP dependent
Mediates 3-stranded intermediate and strand exchange |
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Term
| What happens to cells defective in RecA? |
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Definition
They cannot undergo homologus recombination
Homologus recombination can rescue stalled replication forks, so cells without RecA are very susceptible to DNA damage |
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Term
| Define site specific recombination |
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Definition
Occurs at specific DNA sequences shared by two molecules
Mediated by integrases or recombinases |
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Term
| Give two examples of site-specific recombination |
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Definition
Bacteriophage integrases
VDJ rearrangement in antibody gene diversity |
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Term
| What do many bacteriophages integrate in their genomes via site specific recombination? |
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Definition
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Term
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Definition
| The system that fixes an occasional dimer "doublet" chromosome after recombinational repair |
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Term
| How many antibodies are possible due to site-specific recombination? |
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Definition
3000 possible light chains x 5000 possible heavy chains = 1.5x10^7 possibilities
B cells go on to mutate for even more diversity |
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Term
| What proteins assist in site-specific recombination in B cells, and where do they bind? |
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Definition
| RAG1 and RAG2 (recombination activating genes) bind to specific "recombination signal sequence" (RSS) |
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Term
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Definition
| Recombination that enables the movement of transposable elements (transposons) "jumping genes" |
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Term
| What are the types of transposons? |
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Definition
Insertion sequences
Complex transposons (often carry antibiotic resistance genes in bacteria) |
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Term
| What are the two types of transposition? |
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Definition
Simple or direct: "cut and paste"
Replicative: forms cointegrate intermediate, donor and recipient both end up with copies of the transposon |
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Term
| What are the three types of recombination |
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Definition
1. Homologus
2. Site-specific
3. Transposition |
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