Molecular Techniques SEH

nucleic acid

Complex, acidic macromolecules that contain genetic codes. Made of bound together nucleotides by the phosphate and hydroxyl groups of their OWN sugar

DNA

Deoxyribonucleic acid, Contains genetic information needed by cells to synthesize all the proteins necessary to maintain their structure, carry out their essential functions and replicate to form new cells

RNA

Ribonucleic Acid; Present in both nucleus and the cytoplasm and plays a major role in protein synthesis and serves as the source of genetic information in RNA viruses

Nucleosides

Composed of a ribose sugar and a NITROGEN BASE

Nucleotides

Phosphorylated nucleosides

Endonuclease

Cut DNA strands internally—Restriction enzymes, DNases

Exonuclease

Remove bases from the ends of DNA strands

Codon

3 nucleotides

Gene

An ordered sequence of nucleotides on a chromosome that encodes a specific functional product

Epigenetics

Mitotically & meiotically heritable changes in phenotype not encoded in the genotype; non-sequence specific

What is the Central Dogma and its meaning

• DNA—>RNA—>Protein

• a theory stating that genetic information flows in one direction: from DNA to RNA, and then to proteins

What are the functions of DNA and RNA

• DNA stores the genetic blueprint of living things

  • Transcription template

  • Replication

  • Inheritability

• RNA converts that code into functional protein

  • mRNA, tRNA, rRNA

  • Gene regulation

Name the four types of nitrogen bases found in DNA

• Adenine

• Guanine

• Thymine

• Cytosine

Name the two ring structures that the nitrogen bases are composed of

• Purine

• Pyrimidine

How many hydrogen bonds are between adenine and thymine

Two bonds

How many hydrogen bonds are between guanine and cytosine

Three bonds

Describe where DNA replication begins, what end of the DNA does replication begin

A new DNA strand is polymerized from the 5’ end to the 3’ end (5’ to 3’), reading the parent strand in the 3’ to 5’ direction

What are the lagging and leading strands of DNA

• Lagging strands: Synthesized in the 5’ to 3’ direction, which forces the enzyme to move away from the opening replication fork.

• Leading strands: Synthesized in the 5’ to 3’ direction, following right behind the opening replication fork.

Name the enzymes used in DNA replication. Which enzyme helps “unwind” the DNA? Which enzyme is used to copy the new DNA strand?

• Topoisomerase

• DNA primase

• DNA ligase

  • DNA helicase unwinds DNA

  • DNA polymerase

What are Okazaki fragments

short, newly synthesized DNA pieces formed on the lagging template strand during DNA replication

What does “antiparallel, complementary and semi-conservative” mean for the double helix DNA?

• Antiparallel— Two complementary single stranded nucleic acids 5’ end of one molecule is next to the 3’ end of the other

• Complementary— Describes the order of two antiparallel single-stranded nucleic acids

• Semi-Conservative— Describes DNA replication where one strand is conserved and serves as a template for a new strand

Name the three naturally occurring factors that can be used in recombinant DNA

• Nucleases—cut DNA into pieces for recombining

• Ligases— Paste DNA fragments together

• Plasmids— Carry the recombined DNA into the cell

What is the function of an exonuclease

Remove bases from the ends of DNA strands, increases the fidelity of DNA replication

What are restriction endonucleases

Cut double stranded DNA

How is DNA carried in prokaryotes and eukaryotes

Prokaryotes

• One chromosome

• No nuclear membrane

• Exons only

• Plasmids may be present

Eukaryotes

• Multiple chromosomes

• Discrete nucleus with histone proteins

• Introns & exons

What are the three ways a plasmid can transfer DNA information

• Transformation

• Transduction

• Conjugation

Name three things that DNA and RNA have in common

• 5-carbon sugar

• phosphate group

• nitrogenous base

Name three things that are different between DNA and RNA.

• Ribose sugar in RNA instead of deoxyribose

• Single-stranded RNA not double-stranded

• Uracil in RNA in place of Thymine

What are the differences between DNA replication and RNA replication

DNA replication creates an identical double-stranded copy of an entire DNA molecule, whereas RNA synthesis creates a single-stranded RNA molecule using a DNA template

What enzyme is used to synthesize RNA

RNA polymerase

How much mRNA and rRNA makes up total RNA, respectively?

• mRNA—2.5-5.0% of total RNA

• rRNA—80-90% of total RNA

Name the three structural changes that take place before mRNA is released into the cytoplasm.

• A guanosine cap is added to 5’ end

• String of adenosines called a poly-A-tail is added to 3- end

• Introns are cut out and exons are spliced together

What enzyme is used to help attach codons to tRNA

aminoacyl-tRNA synthetase

What enzyme is used to help attach amino acids to the polypeptide chain to create peptide bonds?

peptidyl transferase

List the three stages that occur during translation of mRNA

• Initiation

• Elongation

• Termination

What is the basic structure of an amino acid and how is the amino acid determined?

• Central carbon

• Amino group

• Carboxyl group

• Side chain (determines the amino acid)

Name the four different protein structures

• Primary

• Secondary

• Tertiary

• Quaternary

Describe the different components and their function found on a gene

• Promoter—Intiation of transcription

• Exons—Code for the amino acid sequence of the protein

• Introns— Noncoding regions that “interrupt” the exon

• Terminator sequence— Specifies the end of the transcription

Name and describe the two types of gene expression.

• Constitutive—Genes are transcribed continuously, Housekeeping genes have products required constantly by the cell

• Inducible—gene products that are required at specific times during the cell cycle or under certain nutritional environmental conditions

Name the four types of epigenetics and describe each one

1) Histone modifications—Controls the accessibility of the chromatin and transcriptional activities inside a cell

2) DNA methylation— occurs mostly on cytosines or adenines, involved regulation of imprinted gene expression and X-chromosome inactivation

3) Chromatin remodeling— ATP dependent process by enzymes that shift nucleosomes, affects transcriptional activity

4) Non-coding RNA— transcribed and processed but not translated (miRNA). Involved in Cell proliferation, Differentiation, and Apoptosis

What type of specimens can be used to isolate nucleic acids

• Blood

• Buffy coat

• BM

• Solid tissue

• Lavage fluids

• Bacteria, viruses

• Fungi

• Organelles, mitochondria

What type of specimens are adequate for PCR amplification

• Dried blood, plasma

• Saliva, urine

• Bone, teeth

• Amniotic fluid, >8 weeks

• Hair follicles, hair shafts

• Buccal cells

• CSF

• Formalin fixed tissue

• Feces

List the four basic steps in nucleic acid isolation

1.) Lysis of the cells and nuclear membranes to release DNA using a detergent

2.) Removal of proteins using “salting out” process, phenol extractions or columns

3.) Precipitation of DNA

4.) Concentration of pure DNA

What are the 3 types of DNA extraction methods and what kind of chemicals are used for each one?

• Organic—uses organic chemicals, phenol, chloroform

• Inorganic—uses inorganic chemicals, detergents, EDTA, acetic acid, salt (salting out, spooling)

• Solid phase—DNA is immobilized on a solid support, beads or columns

When DNA is isolated using organic reagents, what are the two reagents used for extraction?

• Phenol

• Chloroform

What is the preferred reagent when extracting RNA samples

Guanidinium isothiocyanate

What is diethyl pyrocarbonate (DEPC)?

A chemical inhibitor that permanently inactivates Rnases

What are the two extraction methods used for RNA?

• Organic

• Solid phase

How can messenger RNA be isolated?

Using columns covalently attached to polyU or polyT oligomers that hydrogen bond specifically to mRNA polyA tails

Once DNA and/or RNA is isolated, what are the next steps

• Analyze quantity and quality

  • Gel electrophoresis (quality)

  • Spectrophotometry (con., yield, purity)

  • Fluorometry (conc.)

• Properly store them

  • DNA—short term 2°C-25°C, Long term (>1 year), -20°C or -70°C

  • RNA—<30 days, -20°C/-70°C DEPC-treated water

What are the storage requirements for DNA and RNA after extraction

• DNA—short term 2°C-25°C, Long term (>1 year), -20°C or -70°C

• RNA—<30 days, -20°C/-70°C DEPC-treated water or EtOH

What are the desirable ratios for DNA and RNA preparations when using the 260:280 ratio?

• DNA~1.6-2.0

• RNA=2.0-2.3

Which reagent will read at 270-275 nm and cause possible contamination problem

Phenol

What are the standard concentration formulas for DNA and RNA purity at 260 nm

• A₂₆₀ unit of double stranded DNA=50ug/mL

• A₂₆₀ unit of RNA=40ug/mL

What kind of charge does DNA/RNA have, positive or negative?

Negative

Name the four types of electrophoresis that are used in nucleic detection and how they work

1.) Agarose— testing performed horizontal format, detects 100-60,000 base pairs. The number of base pairs to be detected will determine the conc. of agarose used

2.) Polyacrylamide Gel (PAGE)— testing performed in a vertical format, detects 10-1,000 base pairs, the number of base pairs to be detected will determine conc. of acrylamide used

3.) Capillary Gel Electrophoresis (CGE)— used to separate nucleic acids, separates solutes by charge/mass ratio, sample is electrokinetically injected at one end and migrates toward the other electrode, separated components are detected near the end of the capillary, Fluorescent labels attached to nucleic acids

4.) Pulsed Field Gel Electrophoresis (PFGE)— Used for separation of very large DNA molecules, pulses of current is applied to the gel in alternating dimensions to enhance migration, often used in bacterial typing

What is the relationship between matrix concentration and the size of the nucleic acid being detected?

Inverse relationship

List two types of matrices used for gel electrophoresis

• Agarose

• Polyacrylamide

Which type of electrophoresis is best for detecting base pairs ranging from 10-1,000

Polyacrylamide electrophoresis

What type of tracking dye is commonly used in electrophoresis

• Bromophenol Blue

• Glycerol or Ficoll

What type of testing is used in pulse-field gel configuration

Bacterial typing for epidemiological purposes (outbreaks)

List the four types of pulse-field gel configurations used in testing nucleic acid

• FIGE

• TAFE

• CHEF

• RGE

How does capillary electrophoresis separate nucleic acid?

by size using a high-voltage electrical field applied across a narrow, polymer-filled tube

What are the results from capillary electrophoresis displayed on

electropherogram

What are the advantages and disadvantages of using capillary electrophoresis compared to traditional slab gel electrophoresis?

Advantages

• Increased sensitivity

• Immediate detection

• Labor and run times are decreased

• Software programs analyte the results gathered by the detector

Disadvantages

• Instrumentation cost

• Fluorescent labeling of samples

What types of buffers are used for DNA and RNA electrophoresis

• DNA—TBE and TAE

• RNA—Sodium phosphate or MOPS

What types of stains are used to detect bands for DNA and RNA on electrophoresis

• DNA/RNA— SyBr green or GelRed, Silver stain (more sensitive)

• dsDNA— EtBr

What is a molecular weight marker/ladder?

A set of standards that are used for determining the approximate size of a protein or a nucleic acid fragment run on an electrophoresis gel

Describe how fluorometry is performed

Concentration is measured against standards with known DNA/RNA conc.

What type of fluorescing dyes are used for DNA and RNA when fluorometry is used

• DNA— Hoechst 33258 or PicoGreen

• RNA— RiboGreen, SybrGreen II, etc

How are concentrations determined in fluorometry?

measuring the intensity of light emitted by a fluorescent sample and comparing it to a set of known standards

Know the calculations used to determine concentration and yield for DNA/RNA spectrophotometry absorbance readings

• DNA conc.= A₂₆₀ reading * 50ug/mL

• RNA conc.= A₂₆₀ reading * 40ug/mL

Know how to interpret purity of DNA/RNA based on the A260:A280 ratio.

• >2.0—possible contamination of RNA

• <1.6—Possible contamination of protein

Know which type of testing is used for integrity/quality, concentration, yield and purity

• Integrity/quality

  • Gel electrophoresis

  • Capillary electrophoresis

• Concentration

  • Fluorometry

• Yield

  • µg/mL*mL=µg RNA

• Purity

  • Absorbance ratios (260/280 ratios=2.0-2.3)

Define restriction enzymes and state why they are used

• Bacterial enzymes that recognize specific base sequences and break or restrict the DNA polymer at the sugar-phosphate backbone

• Provide a convenient tool for molecular characterization of DNA especially in the analysis of gene rearrangements & in mutation detection. Typically 4-6 bp read in the 5’ to 3’ direction

How many types of restriction enzymes are there

4 main types

Which restriction enzyme is the most common

Type II restriction enzymes

How are fragments resolved after a restriction enzyme treated DNA sample is tested on gel electrophoresis?

• The number of bands indicates the number of restriction sites

• The size of the bands indicates the distance between restriction sites

What is hybridization?

The formation of hydrogen bonds between complementary strands of DNA or RNA

How is hybridization used in molecular diagnostics

Used in the detection of a specific DNA or RNA sequence within a sample

What occurs before hybridization takes place on a DNA sample

Denaturation

What should the probe and target be for hybridization to occur

Probe:

• DNA

• RNA

• Oligonucleotides

Target:

• DNA

• RNA

What is the purpose of the probe used in hybridization

To identify one or more sequences of interest within a large amount of nucleic acid.

Name three types of probes that can exist in hybridization

• DNA probes

• RNA probes

• Protein probes

When a probe is “labeled”, what will occur

Makes the probe visible

What is the target and probe used in Southern Blot

• Target: DNA

• Probe: DNA

What are the steps in Southern Blot

1) DNA purification, fractionation, electrophoresis

2) Denaturation

3) Transfer onto membrane

4) Hybridization with labeled DNA probe

4) Detection

What does the probe determine during the Southern Blot process

Which DNA is seen

What is stringency?

The combination of conditions under which the target is exposed to the probe

Name the four factors that will affect stringency

1) Temperature of hybridization

2) The salt conc. of the hybridization buffer

3) The conc. of the denaturant

4) The length and nature of the probe sequence

What is the definition of melting temperature and what is its role in hybridization

• The temperature at which 50% of a nucleic acid is hybridized to its complementary strand

• Probe binding is done under specific conditions for that probe including the optimal temp. bsed on melting point

Know the calculation for melting temperature.

(#G-C) (4°C)+(#A-T)(2°C)

What will increase stringency?

• Formamide (denaturant) conc.

• Low salt

• Heat

What will happen if stringency is too low? Too high?

Too low:

• Results in more forgiving conditions that allow binding between sequences with some mismatches or lower overall similarity

• Used to find related (homologous) sequences or to allow binding to less perfect targets

Too high:

• Results in highly complementary sequences with few or no mismatches will bind

• Used to detect very specific targets or to wash away weakly bound probes

Name two ways that detection of bound probe is performed in Southern Blot

• Radioactive or chemiluminescent detection

• Chromogenic detection

What is Northern Blot used to detect?

specific RNA molecules such as mRNA

What is Western Blot used to detect?

Specific binding proteins, polyclonal antibodies, or monoclonal antibodies

Describe how the antibody is detected in Western Blot

The first antibody is not labeled, and a labeled secondary antibody to the primary antibody is used. The label us a visible color or florescent molecule

What is Dot Blot and how is it used

• Simplified Southern/northern blot, no gel electrophoresis or transfer

• Confirms the presence or absence of molecules detected by the probe

What are DNA microarrays and how are they used

• Consists of an orderly arrangement of specific gene sequences that have been immobilized onto precise locations of a small solid support such as a glass microscope slide

• Used for simultaneous screening of the entire human genome for a gene of interest by hybridization with nucleic acid from a biological sample.

  • Analysis of gene-expression patterns