Molecular genetic techniques Flashcards
The melting temperature of DNA mainly depends on… ?
GC content (b/c form 3 H-bonds instead of 2 btw AT)
What are phosphodiesterases?
also called nucleases
→cleave phosphodiester bonds btw nucleotides
- DNases if specific for DNA
- RNases if specific for RNA
What is the difference btw endo- and exonucleases?
- endonucleases: able to cleave phosphodiester bonds within the nucleic acid
- exonucleases: only able to cleave terminal phosphodiester bonds of nucleic acid, either cut in 5’→3’ or 3’→5’
What are site-specific DNA methylases?
Where can they be found?
only in bacteria
→ methylate bacterial DNA to distinguish it from foreign DNA
What are bacteriophages?
How do they incorporate their DNA into their hosts?
viruses specific to bacteria
- open bacterial genome
- insert their genome, ends resealed
- either via homologous base pairing, or
- synthesize site-specific proteins that connect to bacterial DNA if no base pairs matching
- bact. DNA linearized out
What are restriction enzymes?
also called restriction endonucleases
excise non-methylated DNA in bacteria as defense mechanisms against bacteriophages, i.e. restrict their growth
DNA sequence:
- blunt ends
- overlapping sticky ends
Explain the mechanism of restriction endonucleases.
cleave both DNA strands at specific 4-8 bp short palindromic sequences, so called restriction sites
→ create either blunt or sticky ends
- blunt ends: smooth ends
- sticky ends: ends are overlapping
How does recombinant DNA technology exactly work?
- restriction enzymes used to excise specific nucleotide sequence from any DNA strand
- inserted into vector
- vector introduced into bacteria DNA
→ DNA naturally amplified
Explain the mechanism of complementary sticky end ligation.
complementary sticky end ligation
native sticky ends are used to insert restriction fragment into vector
- bacterial restriction endonuclease creates complementary sequence on the restriction fragment and the vector
- complementary base pairing btw restriction fragment and vector
- ligated by T4 DNA ligase (hydrolysis of ATP → AMP)
What is homopolymer tailing?
used to ligate blunt ended restriction fragments to vectors (instead of sticky ends)
terminal transferase adds nucleotides to vector DNA and restriction fragment, creating sticky ends
→ then ligated by T4 DNA ligase
What are chimeric molecules?
molecules (i.e. nucleic acids) that contain sequences from two different species
What is a cloning vector?
What are general requirements of cloning vectors?
DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell
- needs ori so it can be replicated in bacterium
- needs selectable markers, so cells containing the vector can be identified
- needs spec. cleavage site for each RE used
NOTE: different vectors used in prokaryotes and eukaryotes
What is the most common type of bacterial vectors?
Characteristics.
plasmids
- small, circular dsDNA molecules carrying genes for antibiotic resistance
- have own ori, replicate independently from bacterial genome
List other bacterial vectors. What are their advantages?
- phages (bacterial viruses): have linear DNA
- cosmids: plasmids containing some viral λ-DNA
- .. artificial chromosomes: bacterial BAC, yeast YAC
→ can carry incr. amount of nucleotides
(YAC can carry largest DNA fragments)
What are viral vectors?
vectors used in eukaryotes
- adenoviral vectors
- retroviral vectors
- lentiviral vectors (group of retroviral vectors)
- adeno-associated viruses (AAV)
NOTE: limited in size of DNA sequences that can be inserted
What is an episome?
genome outside the bacterial genome
→ replicates independetly (e.g. plasmids)
What are genomic libraries?
How are they created?
total genomic DNA from a single organism is stored in vectors
DNA cleaved by REs → inserted into plasmids → plasmids introduced into bacteria
What are cDNA libraries?
How are they created?
DNA complementary to fully transcribed mRNAs (after splicing, does not contain introns), stored in vectors
mRNA is extracted → reverse transcriptase used to produce DNA copies (cDNA) of mRNA → inserted into plasmids → plasmids introduced into bacteria
What is an expression vector?
vector designed for gene expression in cells
→ contains entire gene (cDNA) that codes a specific protein
often contain highly inducible promoters
What is the function of probes in genomic/cDNA libraries?
pieces of DNA or RNA that are either flourescent or labelled w/ radioactive nucleotides
→ recognize complementary sequences in bacterial plasmid on Southern or Northern blots
What is PCR?
How does it work?
polymerase chain reaction
method to amplify a specific sequence of DNA
- dsDNA is heated to 90 °C → denaturation to ssDNA
- cooled down and excess of primers added that anneal to complementary sequences
- thermostable DNA polymerase and dNTPs added, heated up again → polymerase reaction
→ then cycle repeated
Which special polymerase is used for PCR?
Main feature + activites.
Taq polymerase
- thermostable, functioning even at high T (90-95° C)
-
activities:
- __5’→3’ polymerase activity
- 5’→3’ exonuclease activity
- BUT: lacks 3’→5’ exonuclease activity
List some applications of PCR.
amplifies DNA for
-
allele specific PCR
→ used to detect single nucleotide polymorphism (e.g. Leiden mutation) - DNA fingerprinting
- quantitative/real time PCR
- DNA sequencing
What is RFLP?
restriction fragment length polymorphism
type of fingerprinting to compare DNA fragments from 2 individuals
- different restriction sites in both samples, REs cut DNA into fragments of different length
- restriction fragments separated according to their lengths by gel electrophoresis (Southern blot)
→ differences btw both samples can show inherited disorders
What is CNV?
copy-number variation
describes the phenomenon that the # of repetitive sequences varies btw individuals in the human population
→ exploited by DNA fingerprinting
How does the DNA fingerprinting work?
each individual has a distinct no. of repetitive microsatellite sequences (2-5 bps) in their genome = CNV
- when this sequence known, primer can be created + microsatellites amplified by PCR
- 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
What is quantitative real-time PCR?
monitors the amplification DNA molecule during the PCR, not at its end → determined by measuring fluorescence emitted by probe that binds to amplified sequence
- fluorescence initially not detectable due to a fluorescence quencher on probe
- as probe pairs w/ DNA, fluorophore separated from the quenching molecule and fluorescence results
- as DNA segment is exponentially amplified by PCR, fluorescent signal also incr. exponentially
What is the function of pGL3 vector?
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
What is the purpose of gel electrophoresis?
How does it work?
seperates molecules according to size
- sample placed into well of gel, electric field applied
- molecules move through pores in gel towards anode (+ charge)
→ larger molecules move slowest, small molecules move fastest
What is blot transfer?
List different types of blot transfer techniques.
techniques based on electrophoresis
- Southern → detects DNA
- Northern → detects RNA
- Western → detects proteins
- Southwestern → detects protein-DNA interactions
Which types of molecule are detected by Southern blotting?
How does it work?
detects DNA
used to determine how many copies of specific gene in tissue or if any alterations in gene
- DNA digested with restriction endonuclease
- restriction fragments seperated by gel electrophoresis
- DNA denaturated by exposure to NaOH + transferred to nitrocellulose membrane (exact replica of pattern on gel)
- cDNA probe added, hybridizes w/ compl. DNA strands on filter
- exposed to X-ray, reveals hybdrized DNA
lab exam ole ole
Which types of molecule are detected by Northern blotting?
How does it work?
detects RNA
pretty much the same as Southern blotting
Which types of molecule are detected by Western blotting?
How does it work?
How is immunoblotting different?
detects proteins
still the same mechanism …
for immunoblotting antibodies are added to the filter (maybe even antibodies for antibodies), then detected by chemoiluminescence
How does ELISA work?
enzyme-linked immunosorbend assay
uses antibodies and color change to detect presence of antigen in a liquid sample
- sample containing antigen put into multi-well microtiter plate, antigen adheres to surface
- antibody w/ linked reporter enzyme added, binds to antigen
- plate washed, so only antigen w/ antibody-reporter enzyme left
- substrate for reporter enzyme added, reaction yields colorful compound
check out those pretty colors onthis microtiter plate
How do you DNA microarrays work?
slides w/ imprinted collection of cDNA
used to compare gene expression in of cancer/non-cancer cells
- mRNA isolated from both cell populations, then converted by reverse transcriptase to cDNA
- cDNAs are labeled, non-cancer cells: green, cancer cells: red
- cDNAs mixed, hybdrized to cDNA on chip
- 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)
What are transgenic organisms?
in most cases, transgenic mice created
exogenous gene (= transgene) introduced into living organism, so that genetically modified offspring is produced
What does RNA interference do?
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
What are ribozymes?
catalytically active RNA molecules, also used in gene therapy
What are aptamers?
RNA or DNA based protein antagonists that are introduced into host cell and used for gene therapy
What are Zinc finger endonucleases (ZFNs)?
How do they work?
artificially created, dimeric restriction endonucleases
- 2 DNA binding monomers, containing Zinc finger domains, bind to specific dsDNA sequence (9bps)
- nuclease domains bind to DNA binding monomers, cut both DNA strands
What is the (original) function of the CRISPR/Cas9 system?
acquired bacterial defense mechanism against bacteriophage infection
→ RNA-guided endonuclease system that cleaves dsDNA, causing a double strand break preventing virus from replication
Describe the structure and the function of the individual components of CRISPR/Cas9.
- 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
How does CRISPR/Cas9 detect viral DNA?
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)
Why does everyone love CRISPR/Cas9?
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.
What is the purpose chain termination method of Sanger?
method of DNA sequencing
- DNA + RNA pol + normal dNTPs is put into 4 different tubes, each tube containing one type of didNTP (radioactively labeled)
- 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
- gel electrophoresis w/ 4 columns (1 for each type of didNTP) used to seperate fragments
- exposed to X-ray, complementary DNA sequence of DNA sample can be read from bottom to top
How is the nowadays ued, automated method of DNA sequencing different from Sanger’s original method?
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
How does pyrosequencing work?
again, method of DNA sequencing
whenever nucleotide incorporated into synthesized DNA strand that is complementary to sample DNA, PPi 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
Which non-viral vectors are used in gene therapy?
- liposome-based vectors
- modified transposons
