LEC28: Nucleic Acid Analysis

Question 1

what is a DNA oligonucleotide?

what are important structural features?

Answer 1

string of 18-25 ntds that’s complementary to a sequence on the mRNA

serves as a “probe” for an mRNA containing the target sequence

5’ end: radioactive phosphate group or another detectable chemical group

Question 2

what is the purpose of different experimental methods looking at nucleic acid synthesis?

Answer 2

diff experimental methods to probe for transcriptomes or genomes

Question 3

what is the purpose of Northern Blotting?

Answer 3

detect a specific part of an RNA out of all the mRNAs

Question 4

what is the transcriptome?

Answer 4

the whole set of RNAs a cell expresses in a particular situation

different cells have different transcriptomes

Question 5

what kind of RNA is used in northern blotting? why?

Answer 5

cytoplasmic mRNA because has **poly-A tail **which makes it localizable

nuclei are removed to avoid contamination w/ pre-mRNA

Question 6

in northern blotting, how is isolated poly-A+ mRNA treated?

Answer 6

pass it through an **oligo-dT column **

get **A-T complementary base pairing **

**poly-A tail **sticks to oligo-dTs, rest of mRNA is washed away

Question 7

in northern blotting, what happens to the poly-A+mRNA w/ its oligo-dT tail?

Answer 7

load into a **gel, separates by size **

then blot the gel onto a membrane and apply a probe that binds to **target mRNA sequences by complementarity **

wash filter; expose to X-ray film; see band pattern

Question 8

what is the probe in Northern blot?

Answer 8

a single-stranded DNA that’s radioactively labeled at its 5’ end

sequence is complementary to sequence known to be on target mRNA

Question 9

what information do we get from Northern blotting?

Answer 9

1) tissue-specific gene expression
2) regulated expression
3) alternative splicing
4) intsertion/deletion mutations
5) defective splicing

6) quantitative info about level of expression, b/c amount of probe that binds is function of number of target molecules present

Question 10

how can northern blot tell us about tissue specificity?

Answer 10

glucose in liver vs glucose in brain is good example-

the more mRNA in gel, the more probe binds, the more intense the signal is

so see from gel **tissue specificity where diff genes are expressed differentially **based on their location/function

Question 11

what is the purpose of doing PCR?

Answer 11

amplify a specific DNA fragment from a complex mixture

Question 12

what is the order of the first steps of PCR, before amplification?

Answer 12

1) heat mixture to separate DNA strands
2) add 2 specific oligonucleotide primers- complementary to short stretches on eithe rside of desired fragment
3) lower temperature

primers hybridize to DNA at desired ends of target sequence

4) add heat stabled DNA polymerase & nucleotide triphosphates

Question 13

what does heat-stablized DNA polymerase do in PCR?

Answer 13

extends the primers, synthesizes new complementary DNA strands

Question 14

what is produced at end of 1st cycle of PCR?

Answer 14

2 double stranded DNA molecuels containing target sequence

Question 15

how many double stranded DNA molecules at end of 2nd PCR cycle? 3rd?

Answer 15

2nd: 4
3rd: 8

Question 16

describe the process of controlling the PCR tube reaction conditions and what happens at each step?

Answer 16

1) denature at 95 degrees C
2) extension by adding heat-stablized DNA polymerase which extends from 3’ end of primer at 72 degrees C
3) anneal to allow specific primer/target hybridization/binding at 50-60 degrees C

Question 17

what is the basis of PCR?

Answer 17

primer specificity - using an oligo primer w/ a 20-ntd sequence that’s complementary to a known 20-ntd sequence will **only bind to particular sequence of interest **

Question 18

how can we be confident PCR works properly?

Answer 18

use 2 oligos complimentary to sequences known to be located a certain distance from each other

there’s virtually 0% chance that you find anything except for the exact localized pair of ~20-ntd sequences 300 base-pairs apart from each other that you wanted to find

Question 19

how does primer extension work in PCR?

Answer 19

3’ OH is free on oligos

have dNTPs and a polymerase in test tube

polymerase in response to DNA template and 3’ OH of oligos will extend primers in each direction, amplifying # of copies w/ each round of PCR

Question 20

what are the diagnostic uses of PCR?

Answer 20

1) detection of carrier for genetic diseases
2) single nucleotide polymorphisms, SNPs
3) PCR for RNA target

Question 21

how does PCR for point mutations/indels work?

Answer 21

use PCR to amplify a particular chromosomal DNA segment w/ known DNA mutations

Question 22

what is a restriction endonuclease?

Answer 22

enzyme that cuts DNA in middle fo the sequence at a specific target sequence

Question 23

what id Dde I?

Answer 23

a restriction endonuclease

Question 24

when using PCR for detection of a carrier of a genetic disease,

how many bands show up on a gel if the carrier is a heterozygote?

Answer 24

3 bands:

1 for the wildtype, b/c does not have restriction endonuclease’s recognition site

2 bands for where the restriction endonuclease cut the mutated sequence of interest

Question 25

how is PCR used for “foreign” genome detection?

Answer 25

can do PCR for a “foreign” genome, such as TB’s genome, rather than ours, from a sputing sample

can get immediate detection of that foreign organism’s genome’s presence in culture

Question 26

what is real-time PCR?

Answer 26

follow, in real time, course of amplification of target sequence

as number of cycles of PCR reaction continues, get more and more of target DNA present which is detectable by level of flourescence (i.e. of SYBR Green) that flouresces when it has bound to target DNA sequence of interest

rate at which flourescent intensity increases is directly related to quantity of input taget sequence

**how soon (time) sample rises above detection level = signaling mechanism **

Question 27

How can PCR be used to look at RNA? why does this need to happen?

Answer 27

usually, PCR works by DNA polymerase, SO

convert RNA to CDNA, a complementary copy of mRNA sequence via use of reverse transcriptase

Question 28

describe process of using PCR to look at RNA genome expression

describe conersion of RNA –> cDNA

Answer 28

to convert RNA to cDNA:

1) put mRNAs into tube w/ oligo-dT primer, which binds to poly-A tail of mRNAs
2) put oligo-dT bound poly-A+ mRNAs into tube w/ reverse transcriptase & dNTP
3) reverse transcriptase will convert mRNA to cDNA
4) get hybrid between mRNA and cDNA

treat w/ NaOH to destroy RNA

add DNA polymerase, dNTPs

5) now do PCR w/ gene-specific primers to new double-stranded cDNA

Question 29

what is this?

Answer 29

quick detection of a DNA sequence in a tumor cell is possible using real time PCR

see time-related increased flourescence of sequence of interest in tumor versus normal tissue indicates presence of tumor sequences in sample

Question 30

how can SNPs be looked at via PCR?

Answer 30

Next-Gen sequencing

1) purify out RNA
2) bind poly-A+ fraction of mRNA

bind to oligo-dT column

3) take fragments, convert to cDNA
4) do PCR of **target sequence including a SNAP **
5) can analyze for **single nucleotide polymorphisms **in a patients’ genotype w/ respect to the population for a particular polymorphism

Question 31

what does analyzing a person’s SNPs of the ApeE gene tell us?

Answer 31

different polymorphic forms of the ApoE gene have been associated with risk of early onset Alzheimers;

an individual w/ E4 version, if heterozygote, has 4x risk of early onset Alzheimer’s; homozygote = 10x risk

can see this on a PCR

Question 32

what is Tay Sachs caused by?

Answer 32

30%: G-C bp switch in intron 12

70%: 4 bp insert in Exon 11