Genetics Tools Of Molecular Genetics And Cytogenetics

Front 1

What two questions does molecular genetics aim to answer?

Back 1

1. what is the mutation in the genome?
2. How will that mutation creat disease or affect the system?

Front 2

What two fundamental obstacles do geneticists face?

Back 2

1. Obtaining enough DNA or RNA to analyze
2. Purification of the sequence of interest

Front 3

What is the difference between in vivo and in vitro molecular cloning?

Back 3

In vivo= slower "traditional" method, use actual cells to make copies of the DNA of interest. In vitro = faster, DNA cloned mechanically (i.e. PCR)

Front 4

What are some examples of markers?

Back 4

radiometric markers, colormetric, or code for specific properties like resistance to a specific antibiotic.

Front 5

What are the four steps of molecular cloning?

Back 5

1. DNA fragmentation with restriction endonucleases
2. Ligation of DNA fragments to a vector
3. Transfection/ transformation
4. Screening/ selection

Front 6

What is the difference between transfection and transformation?

Back 6

1. Transfection = vector injected into a eukaryotic cell
2. Transformation = vector injected into prokaryotic cell.

Front 7

What are restriction endonucleases?

Back 7

"molecular scissors" used to cut DNA into fragments.

Front 8

What are 4 ways that could be used to isolate a DNA fragment of interest?

Back 8

1. PCR
2. Restriction enzyme digestion
3. DNA sonication and fractionation
4. Chemically synthesized oligonucleotides.

Front 9

What is a common method of molecular cloning?

Back 9

1. restriction endonuclease cuts plasmid.
2. DNA sequence is injected into plasmid along with marker for abx resistance
3. Plasmid injected into bactera.
4. bacteria on antibiotic agar - those that survive have marker.

Front 10

What is one consideration when using markers for molecular cloning.

Back 10

There is no guarantee that cloned DNA with marker ALSO contains DNA of interest.

Front 11

What is a Genetic Library?

Back 11

A collection of clones. Each carries a vector with different DNA fragments. Will theoretically contain all sequences from initial DNA source.

Front 12

What are the 4 types of vectors and their capacity?

Back 12

1. Plasmids - 10 kb
2. Phages - 23kb
3. Cosmids - 45kb
4. YAC (yeast) - 1000kb

Front 13

What is the most common type of vector used for humans and how many base pairs can it contain?

Back 13

YAC - Yeast artificial chromosome - 1 million base pairs (1000kb)

Front 14

What is a cDNA library? Why is it beneficial?

Back 14

cDNA = Complimentary DNA. Produced from fully transcribed mRNA (no introns) so proteins are readily expressed. Con: no information on extrons, regulatory genes, etc.

Front 15

What part on human DNA is not coding?

Back 15

98%

Front 16

What is used to separate segments of RNA or DNA by size?

Back 16

Gel Electrophoresis

Front 17

What is Southern Blotting used for?

Back 17

To check for presence of DNA sequence of interest in a sample.

Front 18

What occurs during Southern Blotting?

Back 18

1. DNA is cleaved into segments by restriction endonucleases.
2. Segments sorted by gel electrophoresis.
3. DNA transferred to membrane soaked in alkaline denaturing solution to break DNA apart, making single stranded DNA
4. Use hybridization probe to find specific sequence
(DNA -neg- binds to membrane - pos- due to ionic difference)

Front 19

How do you screen a genome library?

Back 19

1. Mix in single-stranded DNA complimentary to DNA sequence of interest along with marker (radioactive, color, etc).
2. This will bind, and you'll be able to look at marker to find specific sequence.

Front 20

What type of DNA analysis is commonly used for DNA "fingerprinting"?

Back 20

Southern Blotting

Front 21

In gel electrophoresis, ____ travel faster/farther, and ___ travel slower.

Back 21

1. small fragments
2. larger fragments

Front 22

What is the major difference between Southern and Northern blotting?

Back 22

Southern = DNA
Northern = RNA

Front 23

What is one weakness of Southern Blotting?

Back 23

Difficult to find mutations.

Front 24

What is an ASO (allele-specific oligonucleotide) best used for?

Back 24

Finding DNA with small mutations, maybe only a few basepairs changed.

Front 25

What is the best source of an ASO probe?

Back 25

A synthetic probe

Front 26

What type of probe can distinguish between homozygous, heterozygous, and mutant genotypes?

Back 26

An ASO (allele-specific oligonucleotide) probe.

Front 27

What does Northern blotting analyze?

Back 27

Sequences of RNA

Front 28

What method of analysis has almost completely replaced Northern Blotting?

Back 28

PCR

Front 29

What does PCR do?

Back 29

PCR (Polymerase chain reaction) is a way to multiply DNA exponentially to make make millions of copies for analysis.

Front 30

How does PCR work?

Back 30

1. thermal cycling - heat up DNA to cause it to split
2. Enzymatic replication - DNA polymerase adds nucleotides to both ends.
3. Repeat

Front 31

What are the two main limits to use of PCR?

Back 31

1. availability of enzyme that will tolerate thermal cycling
2. Identification of specific sequence of interest with complimentary primers.

Front 32

Which enzyme is commonly used in PCR for its resistance to thermal cycling?

Back 32

Taq polymerase

Front 33

What is DNA sequence analysis used for?

Back 33

Predicting amino acid sequence that is encoded by a specific gene. Used to design probes, find mutations, etc.

Front 34

What is the most common type of DNA sequence analysis?

Back 34

Sanger sequencing.

Front 35

What steps are involved in Sanger sequencing?

Back 35

1. DNA broken into fragments of various length
2. attach 4 different colrmetric probes (1 for each nucleotide: ATCG)
3. Tag ends of each DNA strand with colormetric probe.
4. Run through tube machine and it sorts them with lenght/color.

Front 36

What is cytogenetics?

Back 36

The study of chromosomes, structures, and how they're inherited.

Front 37

What proporton of live births are affected by cytogenetic mutations? What percent of pregnancies in total?

Back 37

1. 1% of live births
2. 2% of pregnancies over 35
3. >50% of spontaneous abortions due to cytogenetic problem

Front 38

Name some indicators for chromosome analysis.

Back 38

problems of early growth and development in other children, stillbirths/neonatal death, fertility problems, family history of cytogenetic problems, neoplasias, older maternal age.

Front 39

Chromosomal abnormalities include alterations in ____ or ____, and are commonly results of mistakes during ____>

Back 39

1. chromosome number,
2. Chromsome structure
3. mitosis or meiosis

Front 40

What types of things can cause chromosomal abnormalities?

Back 40

radiation, viruses, chemical exposure, advanced maternal age, parental chromosome abnormaliies.

Front 41

How common are chromosomal abnormalities?

Back 41

1/200 newborns

Front 42

What is aneuploidy?

Back 42

improper number of chromosomes

Front 43

What is monosomy?

Back 43

A type of aneuploidy that results in a single copy of that chromosome

Front 44

What is polysomy?

Back 44

A type of aneuploidy where there are 3 or more copies of a gene.

Front 45

What two things can cause aneuploidy?

Back 45

1. non-disjunction,
2. Anaphase lag

Front 46

What is non-disjunction?

Back 46

homologues fail to separate during meiosis. Can occur in meiosis I or II.

Front 47

What is anaphase lag?

Back 47

One chromosome is left out of the new cell due to lag in separation.

Front 48

What types of gametes are formed by non-disjunction

Back 48

monosomic & trisomic

Front 49

What types of gametes are formed by anaphase lag?

Back 49

Normal and monosomic.

Front 50

Which type of aneuploidy is always non-survivable?

Back 50

autosomal Monosomies

Front 51

The long arm of a chromosome is the ___ arm, the short arm is the ___.

Back 51

1. Q arm
2 .P arm

Front 52

What is translocation?

Back 52

Part of a chromosome rosses over with a non-homologous chromosome during meiosis 1

Front 53

What is inversion?

Back 53

During crossover, iece of DNA attaches upside down/ backwards.

Front 54

What is a deletion?

Back 54

Part of the DNA is lost.

Front 55

What is duplication?

Back 55

Results in repeated DNA sequence/

Front 56

An autosomal trisomy that results in Down syndrome.

Back 56

Trisomy 21

Front 57

What symptoms occur with trisomy 21?

Back 57

low-set ears, simian crease in hand, congenital heart defects, intestinal stenosis, predisposition to leukemia, gap between 1 and 2 toes, mental retardation, protruding tongue, umbilical hernia, hypotonia

Front 58

Edwards syndrome is trisomy ___, and is ___ severe than trisomy 21.

Back 58

1. 18
2. more

Front 59

Trisomy 13 results in ____.

Back 59

Patau syndrome = . Severe mental retardation. Life expectancy a few weeks, but can survive years

Front 60

The autosomal polysomies that are compatible with life are:

Back 60

(least likely to survive) 13, 18, and 21 (most likely to survive)

Front 61

A deletion from chromosome 5 resulting in a short arm.

Back 61

"Cri du Chat". severe retardation, rounded face, heart anomalies, distinct cat-like cry.

Front 62

Presence of an extra X-chromosome in male gamete (XXY, XXXY, XXXXY)

Back 62

Kleinfelter's syndrome

Front 63

What are symptoms of Kleinfelter's?

Back 63

Extra X causes feminization - lack of testosterone, testicular shrinking/ atrophy, tall, long appendages, feminine hair distribution, gynecomastia, etc

Front 64

What is Turner's syndrome?

Back 64

Monosomy X or abnormal second X, results in no ovarian development, short stature, webbed neck, sterility, wide chest, heart problems

Front 65

Multiple X females are ___ and typically asymptomatic

Back 65

Common

Front 66

Multiple Y males are ___ and are typically____

Back 66

common, typically taller than average