Molecular genetic techniques Flashcards

Question

What is CNV?

Answer 1

**_copy-number variation_** describes the phenomenon that the # of repetitive sequences varies btw individuals in the human population → exploited by _DNA fingerprinting_

Answer 2

each individual has a **distinct no. of _repetitive microsatellite sequences_** (2-5 bps) in their genome = CNV 1. when this sequence known, primer can be created + _microsatellites amplified by PCR_ 2. _gel electrophoresis_ used to seperate fragments according to size (# of repeats can be calculated) → used for paternity test, forensic medicine etc. _{similar to RFLP, but much less DNA required due to PCR}

Answer 3

monitors the amplification DNA molecule _during the PCR_, not at its end → determined by **measuring fluorescence emitted by probe** that binds to amplified sequence 1. _fluorescence initially not detectable_ due to a **fluorescence quencher** on probe 2. as probe pairs w/ DNA, _**fluorophore** separated from the quenching molecule_ and fluorescence results 3. as DNA segment is exponentially amplified by PCR, _fluorescent signal also incr. exponentially_

Answer 4

_contains **reporter gene coding for luciferase**_ = vector inserted into DNA to assess activity of certain promoter/regulatory response elements * luciferase causes light emission that can be measured * the more light emitted, the more luciferase expressed, the higher the activity of the promoter

Answer 5

**_seperates molecules according to size_** 1. sample placed into well of gel, electric field applied 2. molecules move through pores in gel towards anode (+ charge) → _larger molecules move slowest_, small molecules move fastest

Answer 6

techniques based on electrophoresis * **Southern** → detects DNA * **Northern** → detects RNA * **Western** → detects proteins * **Southwestern** → detects protein-DNA interactions

Answer 7

**_detects DNA_** used to determine how many copies of specific gene in tissue or if any alterations in gene 1. DNA digested with _restriction endonuclease_ 2. restriction fragments seperated by gel electrophoresis 3. DNA denaturated by exposure to NaOH + transferred to _nitrocellulose membrane_ (exact replica of pattern on gel) 4. _cDNA probe_ added, hybridizes w/ compl. DNA strands on filter 5. exposed to _X-ray_, reveals hybdrized DNA _{lab exam ole ole}

Answer 8

**_detects RNA_** pretty much the same as Southern blotting

Answer 9

**detects proteins** still the same mechanism ... for immunoblotting _antibodies are added to the filter_ (maybe even antibodies for antibodies), then detected by **chemoiluminescence**

Answer 10

**_enzyme-linked immunosorbend assay_** uses antibodies and color change to detect presence of antigen in a liquid sample 1. sample containing antigen put into multi-well microtiter plate, antigen adheres to surface 2. antibody w/ linked reporter enzyme added, binds to antigen 3. plate washed, so only antigen w/ antibody-reporter enzyme left 4. substrate for reporter enzyme added, reaction yields colorful compound _{check out those pretty colors onthis microtiter plate}

Answer 11

**_slides w/ imprinted collection of cDNA_** used to compare gene expression in of cancer/non-cancer cells 1. mRNA isolated from both cell populations, then converted by _reverse transcriptase_ to cDNA 2. cDNAs are _labeled_, non-cancer cells: green, cancer cells: red 3. cDNAs mixed, hybdrized to cDNA on chip 4. chip is scanned spot by spot, _fluorescence_ _measured_ * yellow = equal expression in both cells * green = more expression in non-cancer c. * red = more expression in canc. cells | (positions of DNA sequences known)

Answer 12

in most cases, **_transgenic mice_** created exogenous gene (= **transgene**) introduced into living organism, so that genetically modified offspring is produced

Answer 13

gen therapeutical method that introduces **siRNA via vector** into host cell → binds + inactivates mRNA = _silencing_ used in cases of high cholesterol level, hepatitis B, liver tumors

Answer 14

**catalytically active RNA** molecules, also used in gene therapy

Answer 15

**RNA** or **DNA based protein antagonists** that are introduced into host cell and used for gene therapy

Answer 16

artificially created, **dimeric restriction endonucleases** 1. _2 DNA binding monomers_, containing Zinc finger domains, bind to specific dsDNA sequence (9bps) 2. _nuclease domains_ bind to DNA binding monomers, cut both DNA strands

Answer 17

**acquired bacterial defense mechanism against bacteriophage infection** **→** _RNA-guided endonuclease system_ that cleaves dsDNA, causing a double strand break preventing virus from replication

Answer 18

* **crRNA** (CRISPR): 20 nucleotide RNA sequence that recognizes viral DNA * **tracrRNA** (trans-activating): connects crRNA to Cas9 * **Cas9:** RNA-guided endonuclease, cuts viral DNA * optional _DNA repair template_: used for NHEJ/HDR

Answer 19

recognizes 3 nucleotide sites called **PAM** (_protospacer adjacent motif_) downstream of cleavage site on viral DNA → only if matching, then crRNA sequence tries to match to viral DNA (extremly fast)

Answer 20

**sgRNA** (coding for crRNA and trcrRNA), **Cas9** gene + **target DNA sequence** that should be inserted into genome are packed into vector → transfected into target cell CRISPR/Cas9 can then do _gene knockout, gene editing, gene knockin_, etc.

Answer 21

**_method of DNA sequencing_** 1. _DNA + RNA pol + normal dNTPs_ is put into 4 different tubes, each tube containing _one type of didNTP_ (radioactively labeled) 2. since didNTPs **lack OH-group at 3'C**, polymerization is terminated b/c new dNTP cannot bind to it → no continuous, rather fragmented DNA backbones of diff. lengths formed 3. gel electrophoresis w/ 4 columns (1 for each type of didNTP) used to seperate fragments 4. exposed to X-ray, complementary DNA sequence of DNA sample can be read from bottom to top

Answer 22

_does NOT require radioisotopes/X-ray for detection_ 4 differently colored, **fluorescently labelled ddNTPs** are used instead, put into 1 tube → when **excited by laser beam**, wavelength measured by detector, recorded by computer

Answer 23

_again, **method of DNA sequencing**_ whenever _nucleotide incorporated_ into synthesized DNA strand that is complementary to sample DNA, **PP_i released**, used to _reform ATP_ → used by **luciferase** to emit light ⇒ intensity of light shows if more than 1 of these "letters" in a row * only 1 out of 4 dNTPs added/available at a time * previous nucleotide letter is degraded before the next nucleotide letter is added for synthesis ⇒ repeated with each dNTPs until the DNA sequence of the ssDNA template is determined

Answer 24

* liposome-based vectors * modified transposons

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Molecular genetic techniques Flashcards

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