Immunology Molecular Diagnostic Techniques

Nucleic acids carry what?

Genetic information that codes for protein structure

What is the central dogma:

Dna -> RNA -> protein

-Transcription to Translation

A nucleotide is composed of?

1. Phosphate group

2. Pentose sugar

-5' carbon is bound to phosphate grouo

-3' carbon has hydroxyl group (OH)

3. Nitrogenous base

-Attached to 1' carbon of pentose

Whats the main difference between DNA and RNA?

DNA lacks a 2' hydroxyl group

List the Purines

Adenine and Guanine

-Pur As Gold

List the Pyrimidines

Thymine, Uracil, Cytosine

If nucleotides are monomers of DNA what are polymerss called?

Nucleic acids

What is the backbone of DNA called?

Sugar-Phosphate backbone

-Held together by Phosphodiester bonds

-5'-3' polarity

Two DNA nucleotide strands align in which way?

Antiparallel orientation

Strand of DNA are held together by?

Hydrogen bonds

How many hydrogen bonds does A with T have?

2

How many hydrogen bonds does C and G have?

3

What is the overall structure of DNA?

Double helix

What packages DNA?

Histones

-Act like spools around which DNA wraps

-Histone octamer = nucelosome

String of nucleosomes fold further to create?

Chromatin

-Condenses even more to form chromosomes

-Two sister chromatids joined by centromere

What are the 4 stages of the cell cycle?

1. G1 phase

-Cell grows in size

2. Synthesis phase

-DNA is replicated

3. G2 phase

-Cell grows more, checks DNA and prepares for mitosis

4. M phase

-Chromosomes seperated into two daughter cells

Define Semiconservative Replication:

The two strands of DNA separate and each strand serves as a template for a new complementary strand

-Two antiparallel dna molecules with one old and one new strand

In Eukaryotic cells where does DNA replication begin?

Multiple origins of replication

-DNA unwinds forming a replication bubble

-Each bubble has two replication forks moving in oposite directions

List the steps of DNA replication:

1. DNA replication begins at fork where DNA helicase separates two parental strands

2. A short segment of RNA Primer is laid down first by RNA primase

-The primer provides a free 3' hydroxyl grouo

-Will serve as starting point for DNA strand synthesis

3. DNA polymerase then builds the new DNA strand matching bases to template strand

-5'-3' direction

4. DNA ligase seals the breaks in the phosphodiester bond

Describe how the two strands differ in DNA replication:

1. Leading strand

-Continuous 3'-5' fork and moves same direction as helicase

2. Lagging strand

-DNA is made in short discontinuous stretches called Okazaki fragments

What are the 3 types of RNA?

mRNA

tRNA

rRNA

Describe mRNA

1. Messenger RNA

-Single stranded

-Code from DNA template strand is copied with complementary base pairs

-Transcription

2. During transcription mRNA maintains a DNA triplet code in form of codons = code for particular amino acid

3. Translation

=Building a protein from mRNA

Describe Ribosomal RNA:

Structural component of ribosomes

-Where protein synthesis occurs

-Helps translate message from mRNA into protein

Describe Transfer RNA:

Carrier of Amino Acids

-Contain anticodons that allows each tRNA to carry a specific Amino Acid

Describe translation with all RNA:

Anticodon of tRNA will complementary base-pair with codon of mRNA at ribsome

Describe Transcription:

1. DNA strand separated by RNA polymerase

-One serves as template strand

2. RNA polymerase binds to DNA template at promoter regions

-mRNA is synthesized in 5'-3' direction

Describe Translation

1. mRNA processed

2. Ribosomes clamp onto mRNA and read codons

3. Each codon paired with anticodon of tRNA

4. RIbosome links amino acids together with peptide bonds forming a growing protein chain

Define Variants:

1. General term for any sequence change

2. Germ-line encoded (inherited)

3. Can be harmful or benign

Define Mutations:

1. Subset of variants that disrupts gene function

2. Occurs spontaneously in somatic cels

3. Associated with disease

Define Polymorphisms:

1. Sequences changes present in greater than 2% of the population

2. Benign but may influence disease risk or immunity

3. SNPs (single strand polymorphisms) or VNTRs/STRs (variable repeat sequence)

What is Electrophoresis used for?

Study mutations, variants, and polymorphisms

-Detects sequence changes

-Separate fragments of different sizes

-Compare unknown samples to standard

Describe the principle and media of DNA Electrophoresis:

1. Separates DNA fragments by size

2. Gel matrix = Agarose (forms a porous gel)

3. Principle

-Sugar-phosphate backbone of DNA is negatively charged and migrates towards anode (+)

-Smaller fragments move further/faster

-DNA bands seen under uv light

What is the Principle of Capillary Electrophoresis?

1. More sensitive, semi-automated

2. Narrow capillary tubes filled with matrix

-DNA fragments migrate under an electric field

-shorter fragments move faster

-DNA fragments labeled with fluorescent dyes

-Laser excited dyes as fragments pass

-Emitted light captured as electropherogram peaks

Define Restriction Enzymes:

Molecular scissors

-Each one recognizes a very short specific DNA sequence and cuts that DNA out

*CRISPR-Cas9

Describe CRISPR-Cas9:

Used to target the Mrs2 gene by introducing stop codon into gene sequence

*Introduce BamH1 = only edited DNA would be cut by enzyme while unedited type remian uncut

Describe the Hybridization Method:

Involves the binding of two complementary nucleic acids, most notably a template and a probe

*Microarrays

Describe Microarrays:

1. Allow for multiple targets or samples to be analyzed at same time

2. Test sample labeled with fluorescent dye and added to a glass slide containing thousands of specific unlabeled probes

-Fluroescent spots appear where complementary binding occurs

Describe Bead Arrays:

Used for multiplex detection of proteins and nucleic acids

-Tissues typing or respiratory virus panels

-Beads may have antibodies or single stranded oligonucleotides

Describe FISH (fluorescence in Situ Hybridization)

1. Uses fluorescent labeled probes to detect specific DNA sequences

2. Detects chromosome abnormalities

Describe Polymerase Chain Reaction:

Amplifies a specific DNA region defined by two primers, producing millions of copies

What are the three stages of PCR?

1. Denaturation

-DNA is separated into single strands (hydrogen bonds broken by heat)

2. Annealing

-Synthetic DNA primers complementary to the gene region of interest attach

3. Extension

-Thermostable DNA polymerase adds complementary nucleotides to 3' end

*25-35 times in a thermal cycler

Describe Transcription-Based Amplification (TMA)

1. RNA is the target as well as primary product

2. Reverse transcriptase makes a DNA copy of RNA called cDNA

-cDNA;RNA hybrid

3. RNA portion is then degraded by an enzyme called RNase Ii which leaves behind single-stranded DNA (ssDNA)

4. Reverse transcriptase then build a complementary DNA strand using the ssDNA as template to create dsDNA

5. RNA polymerase then is used to create multiple copies of RNA transcript

6. RNA products detected by chemiluminescence

Describe Strand-Displacement Amplification

1. Reaction is isothermal

2. step 1:

-Denaturation (Only do once)

-Seperates template strands

3. Step 2:

-Primers binds

-One primer is special (has restriction enzyme site)

-Other primer is the Bumper primer = binds upstream of first primer

4. Step 3:

-Extension & Displacement

-DNA polymerases bind both primers

-As polymerase move along it BUMPS the New DNA made from first primer off template strand

5. Step 4:

-Nick and Extend

-Strand made from first primer binds to more strands of DNA in solution

-Bumped strand still has restriction enzyme sequence which nicks the strand creating a gap in the sequence

-DNA polymerase builds new strand from gap and bumps off old strand

Describe Branched DNA:

Not target amplification instead signal is amplified

-Target DNA/RNA is captured by probes on a solid surface

-Extender probes attach, then a preamplifer binds creating branches

-Amplifiers attach to branches, each carrying man reporter molecules

What are the results and applications of Branched DNA?

1. Small amount of target nucleic acid = very strong detectable signal

2. Used clinically to detect viruses like HIV, Hep B and Hep C

What is the most definitive method of identifying genetic mutations or polymorphisms?

DNA sequencing (Sanger sequencing)

Describe Sanger (Chain termination) sequencing:

1. Uses all components of PCR plus fluorescent-labeled dideoxynucleotides (ddNTPs) to synthesize DNA complementary to target

-When ddNTPs are added = DNA synthesis stops and rand fragments of varying sizes are produced

Each ddNTP is labeled with?

A different fluorescent dye so the final sequence can be read from order of colors

What is Pyrosequencing?

A DNA sequencing method that detects light produced during DNA synthesis

-It measures chemical reactions that happen as nucleotides are added in real time

Describe the steps of Pyrosequencing:

1. Nucleotide incorporation

-dNTPs are flowed in reaction one at a time in order

-If nucleotide is complementary to template, DNA polymerase adds it to growing strand which releases PPi (pyrophosphate)

2. Signal generation

-Released PPi starts an enzyme cascade

-PPi converted to ATP

-FUels Luciferase reaction that produces light

-Amount of light is proportional to how many nucleotides were added

Describe Next-Generation sequencing:

1. Can sequence large numbers of templates simultaneously

2. Using bioinformatics, the short sequences are assembled to determine the complete sequence which is compared to known databases

For a probe to hybridize to the target, which condition must be met?

The target must have complementary bases.

Process in which two complementary nucleic acid strands bind, such as a DNA probe pairing with a target sequence.

hybridization

Which best describes the principle of microarrays?

The sample is labeled with a fluorescent tag.

Strand displacement amplification shares which characteristic with the polymerase chain reaction?

Both involve DNA polymerase

Sanger chain termination sequencing differs from polymerase chain reaction because:

Dideoxynucleotide triphosphates are added.

What type of signal is generated in pyrosequencing?

Light

Multiple probes bind to target sequences in:

Branched DNA amplification

method that detects target DNA in intact cells

FISH

Next-generation sequencing involves:

generating many short sequences from fragmented DNA.

In which technique is the signal amplified instead of the target DNA?

Branched DNA amp