PCR Flashcards
Why use PCR instead of Ampelography to identify varieties
-Some varieties hard to tell apart
-PCR analyses genome (their genetic fingerprint)
Identifying grapevines genetically with PCR is easy, except for:
Varieties which derived from each other by somatic mutations
Pinot noir and Pinot gris
Define Genome
All genetic material of an organism; DNA (RNA in some viruses)
What is the structure of V. vinifera DNA
Double helix
Double-stranded helical molecule
Where is V. vinifera DNA located
In the nucleus, mitochondria, and chloroplasts of grapevine cells
What are the nucleobases and how to they pair?
Adenine (A)
Cytosine (C)
Guanine (G)
Thymine (T)
Base pairing:
A-T (2 hydrogen bonds)
C-G (3 hydrogen bonds)
Base bairing ensures genetic stability
What is the backbone of V. vinifera DNA (what is it composed of)?
Deoxyribose (sugar) and phosphate
How many chromosomes do V. vinifera have?
19
How many base pairs does V. vinifera DNA have?
about 500 million
What is the direction of V. vinifera DNA
Antiparallel:
One strand runs 5’ → 3’, the other 3’ → 5’
Ensures proper replication and transcription processes in grapevine cells.
How do you check the purity of a DNA sample
With a spectrophotometer by measuring absorbance at specific wavelengths
Key wavelengths:
260 nm → DNA absorbs UV light strongly at 260 nm due to nucleic acids.
A wavelength peak at 260 nm confirms DNA presence. What you want
280 nm → Proteins absorb UV light at 280 nm (mainly due to tryptophan & tyrosine). A wavelength peak at 280 nm suggests protein contamination
230 nm → Presence of organic contaminants (e.g., phenol, salts, carbohydrates). A wavelength peak here is definite contamination
Explain PCR
PCR (Polymerase Chain Reaction): a technique used to amplify specific DNA sequences.
Making millions to billions of copies from a small initial amount.
It is commonly used before DNA sequencing to generate enough DNA for analysis
Explain the PCR three step process (POTENTIAL EXAM QUESTION)
3 step cycle, repeated about 30 times to amplify DNA.
1 cycle = Denaturation → Annealing → Extension
Steps:
1. Denaturation (95°C for 1 minute) – DNA Unwinding:
-DNA heated to break hydrogen bonds
-results in single strand DNA: template for replicating
- Annealing (~60°C) – Primers Bind to DNA:
-Primers = chemically synthesized fragments of DNA
-Temperature is lowered to allow primers to bind to DNA
-primers are starting point for DNA synthesis - Extension (72°C) – DNA Polymerase Builds New Strands:
-The enzyme polymerase extends the DNA strand by adding nucleotides (A, T, C, G) to the primer.
=New complementary DNA strands: doubling the DNA amount in each cycle
Why are primers so important in the annealing step of PCR
They decide which parts of genomic DNA are amplified
They flank the target DNA (forward primer binding in the 5’ direction of target sequence and a reverse primer binding in the 3’ direction)
The unpaired part binds to their complimentary sequence on DNA
Why amplify DNA with PCR
DNA sequencing requireds a large amount of DNA for accurate analysis
It is highly specific and can target a DNA region
Fast and efficient: able to generate bllions of copies in a few hours
What is an SSR marker and what can it be used for?
A Simple Sequence Repeat (SSR) marker (aka microsatallietes)
-a short, repeating DNA sequence
-Mutate very frequently =
Highly variable between varieties
-Mutations make them longer or shorter
SSR markers detect different alleles at microsatallite loci
How are SSR Markers analyzed? Also called DNA fingerprinting
- DNA Extraction
- PCR: DNA Amplification
- Gel or Capillary Electrophoresis
- Data Analysis & Genetic Profiling
What are SSR markers used for in grapevines?
Used for:
-varietal identification
-clone differentiation
-parental analysis
-trait selection for breeding
-conservation of genetic diversity
What are SSR markers used for in humans?
-profiling in forensics
-cancer diagnosis
-paternity testing
-population genetics
Why do scientists like SSR markers?
-No need to know the sequence, just the size of the PRC products
-Easy to compare between labs or with databases
-No reference samples needed
-VIVC database has thousands of varieties cataloged
Define allele, explain what it means for it to be homozygous or heterozygous, and explain what they control:
An allele is a gene found at a specific location (locus) on a chromosome.
Since organisms have two copies of each gene (one from each parent), they can have:
Homozygous alleles → Both copies are the same (e.g., AA or aa).
Heterozygous alleles → Two different copies (e.g., Aa).
Control:
-traits (ex. berry color)
-disease resistance
-aroma compounds
Explain capillary gel electrophoresis
A method to separate PCR products by length
- PCR products are injected into capillary tube filled with gel
- An electric field is applied
- DNA fragments (neg. charged) migrate through gel towards positive electrode
- Short fragments move faster
Flourescent dyes are added to make fragments viewable
A size standart is added for reference
Complete with the complementary strand (POTENTIAL EXAM QUESTION)
3’ ATTCGGCAT 5’
5’ TAAGCCGTA 3’
