MM 11-13

Question 1

Polymerase chain reaction (PCR)

Answer 1

Used to detect small scale mutations on a isolated part of genome.

The DNA is heated and cooled repeatedly, each time allowing for the melting and replication of DNA. In the first step the two strands of the DNA double helix are physically separated at a high temperature in a process called DNA melting. In the second step, primers are annealed. Primers contain sequences complementary to the target region are added provide a specific start and finish markers for the DNA polymerase. In step three, the temperature is lowered and the two DNA strands become templates for DNA polymerase to selectively amplify the target DNA.

Almost all PCR applications use a heat-stable (capable at high temps) DNA polymerase, such as Taq polymerase (from a thermal bacteria). This DNA polymerase enzymatically assembles a new DNA strand from DNA nucleotides, by using single-stranded DNA as a template and DNA primers, which are required for initiation of DNA synthesis.

Heating and cooling is repeated. Each cycle, as PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified.

Then place the DNA in agarose gel to separate by size or charge. This would help identify deletions, insertions, repeats, or some microsattelites. Proceed to sequencing for higher resolution.

Assess 1-500 bases.

Question 2

Sanger DNA Sequencing:

Answer 2

Small scale mutations: Performed after PCR to identify specific bases. Provides the nucleotide sequence. Ideal for SNPs.

Question 3

Real time PCR:

Answer 3

Small scale mutations: To quantify gene expression by assessing quantity of mRNA by florescence.

Question 4

Microarrays and Gene chip

Answer 4

Allows us to examine many mRNA or DNA simultaneously, thereby assessing expression of certain proteins which can indicate the mechanism for a disease. Knowing the regulation of certain proteins helps us diagnose a tumor type, risk of metals, and the best form of treatment.

Chips are made with thousands of microarrays with phosphorescent complimentary mRNA, DNA sequences. Each microarray might contain DNA:

• sequences from each member of a gene family
• sequences from a number of different gene variants (p53 could be mutated in 500 possible ways).
• sequences derived from each gene of the genome.

Question 5

G-band karyotyping

Answer 5

To study structure and number of chromosomes.

Cells are stimulated to divide and stopped in metaphase. Then treated, dyed, and dropped onto microscope slide.

Examines aneuploidy, large translocations, inversions, and large insertion/deletions.

Question 6

FISH - what is it and what is it good for

Answer 6

Fluorescent In Situ Hybridization:

FISH uses fluorescent probes that bind to specific parts of the chromosome with a high degree of sequence complementarity. Large scale- 5 mega bases to full chromosome.

Used to detect the physical location of a gene on an intact chromosome. Can identify rearrangement disorders or insertions/deletions that would go unseen with karyotyping. However, because probes are bind specifically, you need to know what you’re looking for.

Question 7

Array-based comparative genomic hybridization (aCGH)

Answer 7

Large scale- 1-5 megabases.

Scans whole genome. Is thus equivalent to performing hundred-thousands of FISH tests. Performed for anyone suspected of having a chromosomal abnormality, even after a normal karyotyping.

Picks up submicroscopic deletions, CNV’s, Duplications.

Question 8

Western Blotting purpose and steps:

Answer 8

Electrophoresis used for separation of proteins based on size and charge, used to detect specific proteins in a sample of tissue homogenate or extract.

Step 1: Electrophoresis. Place gel in a tray, normally acrylamide. Samples are applied to wells on top of the gel. An electrical current is established. Proteins will move towards positive node.

Step 2: Transfer the separated proteins onto a membrane.

Step 3: Use a probe to detect a specific fragment. A modified antibody which is linked to a reporter enzyme is added to the protein of interest. When exposed to an appropriate substrate, this enzyme drives a colorimetric reaction and produces a color.

Question 9

Gel Electrophoresis

Answer 9

Electrophoresis is the basis for a number of analytical techniques used for separating macromolecules by size, charge, or binding affinity. DNA, RNA, protein.

DNA has negative charge, so will will move towards positive charge. Slow fragments move faster/further.

Question 10

Nucleic Acid Hybridization

Answer 10

is a phenomenon in which single-stranded DNA or RNA molecules anneal to complementary strands. Though a double-stranded DNA sequence is generally stable under physiological conditions, changing these conditions in the laboratory (raising temp) will cause the molecules to separate into single strands. Lowering the surrounding temperature allows the single-stranded molecules to anneal or “hybridize” to each other.

A variety of different methods use hybridization to detect a particular sequence on a DNA sample. Array technology, Gene chips, Next generation sequencing, Karyotyping, FISH, Chromosomal microarray, Comparative genomic hybridization.

Question 11

Snow Drop (blots)

Answer 11

Southern Dna
Northern Rna
O
Western Protein

Question 12

How to extract info from an electrophoresis gel smear

Answer 12

Hybridization method. Use a probe.

Question 13

Types of electrophoresis gel

Answer 13

Agarose, acrylamide

Question 14

Taq polymerase

Answer 14

A heat-stable polymerase that from a thermal bacteria that is not denatured at high temps. Immune to the cyclic heating and cooling of PCR.

Taq polymerase will copy segments of DNA, which are marked by a specific primer, for application of the DNA.

DNA can then undergo Southern blotting electrophoresis and sequencing to visualize the amplified DNA.

Question 15

Requirements for PCR

Answer 15

• Starting DNA
• Heat stable DNA polymerase (Taq polymerase)
• Specific target primers
• dNTPs (delay nucleotide tri phosphates)

Question 16

DNA sequencing

Answer 16

The ultimate resolution in analyzing DNA is by the sequencing of each base.

Question 17

G banding Karyotyping reveals:

Answer 17

Chromosomal aberrations:
•	Abnormalities of chromosome number 
•	Aneuploidy – Trisomy or Monosomy
•	Translocations
•	Large deletions (greater than 10 bp)
•	Duplications
•	Inversions
•	Ring chromosomes

Question 18

Karyotyping- dark vs light bands

Answer 18

Dark bands are denser, therefor contain fewer genes.

Light band are less dense, therefore contain more genes.

Question 19

Copy number variants

Answer 19

CNV is the phenomenon in which sections of the genome are repeated and the number of repeats in the genome varies between individuals in the human population.

CNVs can be short or long repeats. Short repeats include mainly bi or try nucleotide repeats while long repeats include repeats of entire genes.

Can effect gene expression. Can lead to cancer or disease.

Observed by Array-based comparative genomic hybridization

Question 20

Clinical applications for aCGH

Answer 20

Down syndrome - extra copy of chromosome 21.

Cancers - CNVs may contribute to loss of tumor suppressor genes or increase of oncogenes.

Unexplained developmental delay.

Autism spectrum disorder - fragile X.

Question 21

FISH steps

Answer 21

• First cells are treated to rest in metaphase and made to swell, chromosomes are fixed onto the slide
• Next the chromosomal DNA is denatured so that it will separate into single strands
• Then the DNA probes are flooded onto the slide, which are small pieces of single stranded DNA with a sequence of the gene of interest
• Probes can only hybridize with their complementary sequence on the chromosome
• Excess probes are washed away
• And other probes are attached
• The probes are fluorescent
• The slide is then viewed with a fluorescent microscope.

Question 22

Genome Wide Association Studies

Answer 22

A GWAS is an examination of a genome-wide set of genetic variants in different individuals to see if any variant is associated with a trait. GWASs typically focus on associations between single-nucleotide polymorphisms (SNPs) and traits like major human diseases.