Various Competition Shite

Question 1

What are 3 diagnostic methods for Fragile X syndrome?

(old one, screening one, and most appropriate/modern one)

Answer 1

• cytogenetic exam - cell culturing on folate poor substrate –> “break point” in X
• PCR - for screening
• Southern Blot - most appropriate

Question 2

What is the mutation in SCID-X1 and its consequence?

Answer 2

mutated common gamma-chain gene

no Rs for ILs (2, 4, 7, 9, 15, 21)

no T/NK cells

Question 3

What is the mutation in JAK3 defic. and its consequence?

Answer 3

JAK3 mutation (duh)

defective IL signaling (same #s as SCID)

no T/NK cells

Question 4

What immunodefic. is caused by a mutation in purine salvage enzyme and what are its consequences?

Answer 4

Adenosine Deaminase Defic.

ADA gene

no T or B cells, may have NK cells or may not

Question 5

What is ICF syndrome (its mutation and consequences)?

Answer 5

Immunodefic., Centromeric instability, Facial Dysmorphia

DNMT3B mutation -> methylation issues

agammaglobulinemia and centromeric instability (Chr. 1, 9, 16)

(facial issues + retardation)

Question 6

What molecular genetic method is used for creation of recombinant drugs?

Briefly describe it.

Answer 6

Recombinant Gene Cloning

• foreign DNA is inserted into a plasmid vector after cutting it with a restriction endonuclease
• insertion of the vector into a bacterial cell results in replication of foreign DNA and synthesis of its protein upon subsequent host bacterial cell replication

Question 7

What kinds of drugs are made via recombinant gene tech?

(kind of dumb, just a list)

Answer 7

• polypeptide hormones (insulin)
• vaccine antigens
• Abs
• cytokines
• GFs
• coag. factors

Question 8

What are expression vectors + their features/characteristics (6) ?

Answer 8

Vectors specially engineered for optimized gene expression in their host

1. No intron in expressed gene
2. Optimized ORF (open reading frame, continuous codon stretch without stop codons)
3. Protein Tags - to help in purification
4. Optimized Enhancer/Promoter/Terminator
5. Selection Markers - confer artifical selection trait (abx resistance)
6. Origin of Replication - not special, in all vectors

Question 9

What is a reporter gene?

Answer 9

a gene artificially attached (via recombinant tech) to the regulatory sequence of another gene of interest

convey an easily identified/measured characteristic which is used as a sign of expression of the gene of interest

ex: green fluoro-protein or B-galactosidase

Question 10

what are the advantages of capillary electrophoresis over horizontal electrophoresis? (3)

Answer 10

1. Better capacity - dozens of samples at same time
2. Different gel matrixes - can use diff gels > separate diff molecules (RNA, DNA, protein)
3. More specific separation - 1 bp size differences can be separated

Question 11

What are VNTRs?

What are the two kinds?

Where can they be found + how are they inherited?

Answer 11

Variable Number of Tandem Repeats

can me minisatellite (10-60 bp) or micro- (1-4 bp)

found in coding or non-coding regions

inherited co-dominantly

Question 12

Describe the steps of Sanger sequencing.

Answer 12

1. PCR with dNTPs and fluoro-labelled ddNTPs (no 3’ OH)
2. Chain Termination - ddNTP incorp. stops synth > diff. chain lengths
3. Capillary Electrophoresis - separate diff lengths
4. Laser Reading - reads color of fluorolabel on ddNTP at end of each diff fragment, corresponding to 1 of the 4 G/A/T/C nucleotides

Question 13

What is Sanger sequencing used for generally?

What length of fragments can it handle?

Answer 13

looks for sequence differences in known genes (not for specific known mutations/polymorphisms)

handles 700-1000 bp segments

Question 14

How can heterozygosity vs. homozygosity be determined via Sanger?

Answer 14

if someone is heterozygous for a certain nucleotide locus, that locus will have 2 lower peaks of different color at that locus on their “chromatogram” (graph showing color peaks at each nucleotide locus indicative of which nucleotide is present there)

Question 15

Describe the steps of pyrosequencing (a faster, higher capacity next gen sequencing technique)

Answer 15

1. Primer Hybridization - to template DNA
2. dNTP incorporation - one of the 4 dNTPs added to mixture at a time, incorporation releases PPi
3. Light Generation/Detection - PPi release generates light via luciferase
4. dNTP degradation - apyrase degrades unused dNTPs and next dNTP is added
5. Pyrogram - graph shows if light was released during presence of each nucleotide, showing sequence (if peak is higher, means >1 of that nucleotide in a row in sequence)

Question 16

What are Mendel’s laws?

Answer 16

1. Law of Segregation - 2 alleles for each gene separate during gamete formation
2. of Independent Assortment - alleles of genes on non-homologous chromosomes assort independently during gamete formation
3. of Dominance - recessive alleles are always masked by dominant ones

Question 17

How do class I and II transposons differ?

Answer 17

• Class I - Retrotransposons - “copy and paste”, are transcribed from DNA>RNA then reverse transcribed to be inserted elsewhere (w/ RTase encoded by transposon itself); include LTR/LINE/SINEs; (most transposons)
• Class II - DNA Transposons - “cut and paste”, moved without RNA intermediates by transposases, which make sticky-end DS cut and ligate transposon to target (<2% of genome)

Question 18

What process allows there to be >1 protein product from a single gene?

How does it occur?

Example?

Answer 18

Alternative Splicing

usually via exon skipping carried out by “trans-acting” proteins binding “cis-acting” elements on primary mRNA transcripts to promote/reduce use of a splice site

calcitonin gene makes calcitonin in thyroid / CGRP in hypothal.

Question 19

What are 7 “features” of the genetic code?

(sorry)

Answer 19

1. Triplets/Codons - code for AAs / stop
2. Degenerated/Redundant - 64 codons for only 20 AAs
3. Non-overlapping - each nucleotide belongs to just 1 codon
4. “Comma-Free” - coding regions are continuous, no stops
5. Universal - codons have same meaning in diff. species
6. Wobble Effect - 3rd codon position can be read by diff. bases on tRNA > don’t need 61 diff tRNA for 61 codons
7. Unambiguous - 1 codon codes for 1 AA only

Question 20

What is exome sequencing?

Disadvantages / advantages?

Answer 20

sequencing of only protein coding regions of DNA (~ 30 Mb, or 1% of genome)

85% monogenic diseases are in exome

but in complex disease, most variations are outside exome

Question 21

What is the main method for studying gene expression with PCR?

How does it work?

Answer 21

RT-PCR (reverse transcriptase)

analyzes mRNA expression by using an oligo-dThymidine (15-25 b) to complement the mRNA polyA tail as a primer

then reverse transcriptase makes cDNA (more stable than mRNA) from the mRNA + it is visualized with capillary electrophoresis

Question 22

How does quantitative PCR work?

Answer 22

1. “Taqman probe” with fluorescein at one end, quencher at other is hybridized to DNA in question (when f + q are close, no fluoresence)
2. Primer anneals and extension via polymerase proceeds
3. Polymerase degrades probe, separating f + q -> fluorescence
4. Detection of fluoresecence shows a geometric increase corresponding to exponential increase of DNA product

Question 23

How is qualitative (yes/no, presence/absence) information obtained from qPCR?

and quantitative information?

Answer 23

• if sample is positive for the DNA in question a “suprathreshold fluorescence” is detected after some # of PCR cycles
• the quantity or “copy number” of DNA in sample is proportional to the number of PCR cycles needed before detection of fluorescence is possible (C_T or “cycle threshold”)

Question 24

What is an example of how qPCR can be used in follow-up of treatment of a hematological disorder?

And what method can be used to guide personalized therapy of the same disease?

Answer 24

qPCR can be used to detect levels of BCR-ABL transcripts in CML patients after tyrosine kinase inhibitor therapy (imatinib, etc.)

sequencing can be used to look for specific mutations of the BCR-ABL gene (ex: T315I mutation is resistant to most mAb therapies, needs a special kind, “ponatinib”)

Question 25

How does melting curve analysis work in the use of qPCR for genotyping?

(less important: example of a mutation this can detect)

Answer 25

• PCR is used to amplify the DNA, then it is stained with a fluoro-dye called SBYR that only fluoresces when bound to dsDNA
• gradual re-heating melts the amplified dsDNA and the loss of fluorescence can be measured, indicating the melting point
• normal + mutant alleles have diff. sequences + thus diff. melting points -> presence/absence of mutation can be detected
• (Methylene H4F Reductase mutation can be detected)