Exam 3: Molecular Genetic Analysis and Biotechnology II

Question 1

Sanger’s dideoxy-sequencing method (ddNTP) with gel electrophoresis

Answer 1

ddNTP lacks a 3’OH group which terminates DNA synthesis and terminates extension of the DNA fragment; terminates at diff positions on diff strands which generate a set of DNA fragments of various lengths, each ending in a ddNTP w/ the same base. seq obtained is the complement of the orig template strand (base seq can be determined)

Question 2

Sanger sequencing with fluorescent labels and capillary electrophoresis

Answer 2

each of the 4 ddNTPs is tagged with a diff fluorescent dye; the fragments that end in the same base have the same colored dye attached. products are denatured and fluorescent dye is detected by a laser beam. color of the peaks indicates which base is present

Question 3

types of next-generation sequencing technologies microtechnology and computing capacity (3)

Answer 3

1. illumina sequencing
2. pyrosequencing
3. third-generation sequencing (nanopore)

Question 4

illumina sequencing

Answer 4

millions of oligos line the flow cell and a complementary DNA seq attaches and bends over and attaches to a second oligo forming a bridge. a polymerase synthesizes the reverse strand and they release and straighten. tagged nucleotides are added and fluoresce (reliable and automated, standard for many conditions including cancers of all types)

Question 5

pyrosequencing

Answer 5

allows sequencing of a single strand of DNA by synthesizing the complementary strand one base pair at a time, and detecting which base was actually added at each step. chemiluminescent signals form complementary nucleotide added allows to determine sequence of template (not that reliable but being improved. coming online and reducing costs)

Question 6

3rd generation sequencing: nanopore sequencing

Answer 6

sequence determination of single molecules of nucleic acids that electrophoretically pass through nanoscale pores (cheap and not very accurate)

Question 7

metagenomics

Answer 7

inexpensive sequencing tools (illumina) coupled with powerful programs can determine which types of microbes may be present in a microbiome (related to human health when we evaluate organisms we share our space with)

Question 8

personalized/precision medicine

Answer 8

(metagenomics) how valuable is exome sequencing? analysis of whole exome suggest identify genetic cause of rare diseases in children

Question 9

explain: microbes contribute more genes responsible for human survival than human’s own genes. it is estimated that bacterial protein-coding genes are 360x more abundant than human genes

Answer 9

value of the bacteria is important to human health as we have dependence on these bacteria for good/bad health. # of genes important to own health far exceeds the # in our own genomes

Question 10

the microbiome in IBD: current status and future directions

Answer 10

assess the value of the microbiome (ex IBD) microbiome changes in attempt to restore equilibrium and balance the good bacteria to help improve colon health; studies with changing the composition of gut microbiota

Question 11

molecular techniques that are used to analyze gene function (5)

Answer 11

1. site-directed mutagenesis
2. transgenic animals
3. KO mice
4. silencing genes with RNAi
5. CRISPR/cas9

Question 12

site-directed mutagenesis

Answer 12

study gene function when appropriate restriction sites are not available; modified sequence made chemically (primer) and then inserted into a cloning vector for amplification

Question 13

transgenic animals

Answer 13

organism permanently altered by the addition of a DNA sequence to its genome (knock in or knock out)

Question 14

transgene

Answer 14

green fluorescent protein derived from jellyfish (marker and starting point to modify genomes

Question 15

KO mice

Answer 15

KO of target gene produces no functional copy of the gene and phenotype of the KO organism reveals the function of the gene

Question 16

humanized mouse

Answer 16

is a mouse carrying functioning human genes, cells, tissues, and/or organs. use tools, like CRISPR, to make specific targeted modifications to animal genomes to make them better animal models

Question 17

humanized mice are commonly used as models in

Answer 17

biological and medical research; test drugs and study of human-specific infectious agents (immune sys).

Question 18

gene editing

Answer 18

sequence of the human genome can be precisely manipulated to achieve a therapeutic effect. biggest problem is delivering the modifications to the specific tissues if you want to modify them; easier if target is in the blood bc it’s more accessible

Question 19

types of delivery tools

Answer 19

• naked DNA/RNA
• packaged into a virus
• lipid nanoparticle

Question 20

tools used to modify sequences: siRNA

Answer 20

short double stranded sequences; target genes to knockout a target gene ex oncogene or inappropriately turned on gene

Question 21

tools used to modify sequences: application RNAi (RNA interference) in human disease

Answer 21

treatment of human disease like high cholesterol. RNAi has been shown to reduce the levels of ApoB and blood cholesterol in nonhuman primates

Question 22

tools used to modify sequences: shRNA

Answer 22

short hairpin RNA artificial RNA molecule that pairs back on itself and forms a tight hairpin turn for silencing target gene expression

Question 23

advantage of shRNA

Answer 23

gets produced over and over again so it is more powerful (insert a plasmid that has a promoter attached to a sequence of interest and produces RNA molecule similar to siRNA in situ)

Question 24

designer proteins

Answer 24

creating proteins that have DNA recognition sequences that targets specific parts of the genome; modifying binding proteins so they will recog a sequence we want them to

Question 25

disadvantage to designer proteins

Answer 25

shuffling aas so they bind to specific sequences of dna is hard to do and inefficient because have to figure out what aa change is needed and folding

Question 26

designer oligonucleotides

Answer 26

CRISPR uses nucleic acid targeting system; create right set of nucleic acids in CRISPR to recog its target seq to cut and inactivate it (simple and can do virtually w/ any sequence in human genome)

Question 27

the CRISPR/cas9 sys is a _ immune sys. by delivering the cas9 protein and appropriate guide RNAs into a cell, the target organism’s genome can be _

Answer 27

prok immune sys; can be cut at any desired location (guide or insertion sequence into cas9 tool causes piece of DNA to be inserted and repaired which changes the gene via inactivate mutation or correct the sequence of target gene)

Question 28

ethical dilemma with CRISPR

Answer 28

where does it stop (gene editing)? how much modifications do we allow?

Question 29

the national institute of health’s new somatic cell genome editing will award funds to researchers working to solve problems that will help accelerate research across the field, like _

Answer 29

developing better delivery mechs or better methods for gene editing (translation of these technologies to the clinic for treatment of as many genetic diseases as possible)

Question 30

prime editing

Answer 30

combines 2 key proteins and a new RNA to make targeted insertions, deletions, and all possible single-letter changes in the DNA of human cells

Question 31

CRISPR use in epigenetics

Answer 31

CRISPR used to modify epigenetic changes and recreate pluripotency in cells that we want to reactivate

Question 32

the combination of cas9, guide RNA, and repair template DNA can induce

Answer 32

precise gene editing and the correction of genetic diseases in adult mammals

Question 33

t/f: CRISPR trials are about to begin in people, but we still do not know how it well it works in monekys

Answer 33

true; monkeys studies look encouraging but show there’s still a lot to learn about the gene editing technology

Question 34

gene therapy net

Answer 34

NIH supported resource to get information of gene editing trials that are on the way

Question 35

con with CRISPR

Answer 35

still off target effects with it