Genetic Methods 2

Question 1

What is sensitivity

Answer 1

Ability to find the things you are looking for
Categorizing true positves as positve (finding a needle in a haystack)

Question 2

What is specificity

Answer 2

Ability to distinguish between what you are and are not looking for
Close resemblance but distinct features
“Differentiating between hay and needles)

Question 3

What is the resolution

Answer 3

How much/many details you can examine what kinds of changes or variations you can see

Nucleotide vs chromsome level, single nucleotide locus vs whole genome

Question 4

What are components requried for sanger sequencing

Answer 4

dNTPS
ddNTPS
Taq Polymerase
Primer
Buffer + MgCl2

Question 5

What are ddNTPs

Answer 5

Dideoxynucleotides the special nucleotides used in Sanger sequencing to stop DNA synthesis
Modified versions of normal nucleotides and are missing the 3’ OH group so they cannot add another nucleotide after them and cause chain termination

Question 6

What are the four recation tubes DNA is split into

Answer 6

ddATP ddGTP ddTTP ddCTP

Question 7

What types of products are created through PCR

Answer 7

All possible lengths

Question 8

What is important when you wnat to retrieve the orginal sequence

Answer 8

Sanger is reading the new template that is being made so if you want to get the orignal strand you would have to read the compliment

Question 9

What happens when you label the sequences with dye

Answer 9

Amplicons of all possible lengths are created through PCR amplification and ddNTP chain termination

Question 10

What can seperate the amplicons in sanger sequencing and what detects the flourescence

Answer 10

Capillary electrophoresis will separate the products by size chromatogram detects fluorescence of chain terminating ddNTPs as amplicons migrate through capillary

Question 11

What are the results looking like from Sanger sequencing

Answer 11

Usually a template on where each base bare is on that strand because we are able to detect the length of how far it is and using the colour which base

Question 12

How many primers does sanger sequencing use

Answer 12

Only one amplication of one way two primers would be both ways so you have template and nancent strand

Question 13

What are limits and challenges to sanger sequencing

Answer 13

Can only sequence realatively short DNA fragments (300-1000) non specific primer binding
Formation of single stranded DNA secondary structures leading to sequence artiacts

Question 14

What is a microsatellite

Answer 14

Short tandem repeat (N)n (NN)n (NNN)n

Sequence of DNA that is repeated usually between 5-50 times
found in both coding and non coding sequences
Multiallelic so that short tandem repeats can occur for different lengths

Question 15

What are examples of trinucleotide repat disorders

Answer 15

Fragile X disorder (CGG repeats on the long arm of the X chromosome)
230-4000 repeats in the gene of affected individuals

Huntington’s disease (CAG repeat expansion)

Friedreich’s ataxia (GAA repeats)

Question 16

What are microsatellites as biological markers

Answer 16

Markers of genetic disease
Genetic Variation in populations
Genome mapping

Question 17

How are microsatellite alleles amplified and visualized

Answer 17

Microsatellites are amplified by PCR using primers that flank the repeat region. PCR copies each allele (which differ in length based on the number of repeats). The PCR products are then separated by gel or capillary electorphoresis to determine allele sizes.

Question 18

What will a heteroygous microsatellite look like on a gel

Answer 18

Two bands of different sizes (two allele lengths)

Question 19

What are minisatellites

Answer 19

A variabe number tandem repeat consisting of DNA repeat unit 10-100bp long repeated 5-50 times

Question 20

Where are minsatelitles usually found

Answer 20

Mainly in non-coding DNA part of chromsomal telomeres, protecting ends of chromsome from degradation and centromeres

Question 21

What are the compnents of minisatellite repeat unit

Answer 21

Each unit has a conserved core region flanked by variable regions

Question 22

What have minisatellites been implicated as

Answer 22

Regulators of gene expression

Question 23

What are minisatellites used for

Answer 23

DNA fingerprinting, forensics, paternity testing and studying genetic variation

Question 24

How are minsatellites detected

Answer 24

Minisatellites are too large to amplify by PCR so they are analyzed using
1. Restriction enzymes that cut outside the repeat
2. Southern blotting with readiolabled probe to the core sequence

Question 25

How would you read a parternity test

Answer 25

Each band needs to match up with at least one parent so you can identify the differences

Question 26

What is the key difference between minisatellites and mcirosatelites

Answer 26

Microsatelties: short repeates, detected by PCR Minisatellites: long repeates dected by RE digestion +southern blot

Question 27

Are VNTR multi-allelic

Answer 27

Yes and heterozygous

Question 28

What stain do you use with PCR and gel elctrophoresis

Answer 28

EtBR or SYBR green intercalating agents

Question 29

What does PASA detect

Answer 29

Known single-base changes (SNPs) and small known mutations

Question 30

How does PASA differentiate alleles

Answer 30

It uses allele specific forward primers whose 3' ends perfectly match only one allele

Question 31

How many primers and tubes does PASA use

Answer 31

3 Primer and 2 reaction tubes

Question 32

What would be reacation tube 1

Answer 32

Forward primer A perfect match to WT allele a reverse primer

Question 33

What is in reacation Tube 2

Answer 33

Forward primer B (perfect match to variant allele) reverse primer

Question 34

What will you see in reaction tube 1

Answer 34

This is the forward primer designed perfectly to the WT so if you have the WT then you will have a perfect complementarity and have replication of DNA and if it is a polymorphism mismatch you will have no replication

Question 35

What is in reaction tube 2

Answer 35

This is the primer designed to perfectly complement the variant, so if you have the mutation, then you will have replication, and then the mismatch will have no replication

Question 36

What would you see in the different electrophoresis

Answer 36

Homozygous, see it in both tubes then it is heterozygous

Question 37

What does Bi-pasa detect

Answer 37

Same as PASA known SNPs with better resoultion

Question 38

How many primers does Bi-pasa use

Answer 38

4 primers two allele specifc primer + two outer primers

Question 39

How many PCR reacations are needed

Answer 39

Only one reacation tube

Question 40

How does Bi-PASA work

Answer 40

Each allele- specific primer creates a product only if it perfectly mataches the SNP __> different amplicons form depending on the genotype

Question 41

What does the gel look like in Bi-PASA

Answer 41

You have different primer on each straond so could be PQ PB AQ